Methods of preparing a primary cell sample

ABSTRACT

The invention provides methods of preparing a sample of viable diseased cells obtained from a human subject for clinical testing, wherein the methods inhibit anoikis and/or anoikis in the cells while maintaining the physiological functions and genomic composition of the cells when they were in vivo. In the methods of the invention, primary cells are cultured in media comprising at least one anoikis inhibitor, preferably at least one inhibitor of an intrinsic anoikis pathway and at least one inhibitor of an extrinsic anoikis pathway, under anti-anoikis atmospheric conditions, such as greater than 2% and less than 20% oxygen. Method combining multiple culturing conditions, including surface attachment under conditions that inhibit anoikis, are also provided. Compositions and kits for use in the methods of the invention are also provided.

RELATED APPLICATIONS

This application is a continuation of International Application No.PCT/US2016/057923, filed on Oct. 20, 2016, which claims priority to, andthe benefit of, U.S. Provisional Application No. 62/243,765, filed onOct. 20, 2015. The entire contents of the aforementioned applications isincorporated herein by reference.

BACKGROUND OF THE INVENTION

A number of constraints make it difficult to obtain viable humandiseased cells suitable for use as a sample in a clinical test when thecells are obtained directly from a human tissue specimen. This largelyexplains why there are few, if any, clinical tests that use live humancells extracted from tissue. Oftentimes, the preparation of a viablediseased cell sample must be performed at a different location thanwhere the specimen is removed from a patient. This requires that thetissue be transported in a collection container that retains cellviability for an extended period of time. In addition, the amount ofdiseased tissue available for testing is limited. When the specimen isobtained during a biopsy procedure, 5-10 milligrams or less of diseasedtissue may be all that is available. Conventional techniques to extractviable cells from a tissue specimen of this size yield few viable cells.A clinical test requires more than a few viable diseased cells, whichwould require that the extracted cells to be expanded to have a suitablenumber representing the tumor of the patient. This presents severaladditional constraints. First, the time available to expand the cells islimited. Clinical test results are ideally available within several daysfollowing the acquisition of the patient specimen to ensure timelyselection of an appropriate treatment protocol. In rare cases, aclinical result may still be useful 30 days after a specimen is obtainedfrom a patient. In most cases, the clinical test must be designed toyield a result in less than two weeks to be useful. This requires,ideally, methods to extract and expand a small number of viable cellsextracted from a specimen capable of providing a sufficient number ofviable diseased cells in less than two weeks.

Furthermore, for the clinical test results to be reliable, the cellsmust be extracted and expanded in a manner that maintains thedistribution of diseased cells found originally in the specimen. This isbecause any clinical test involving a human specimen requires that atest sample derived from the specimen be representative of the originalspecimen itself. For instance, a tumor specimen is comprised ofdifferent epithelial cell types—luminal, myo, basal, stem—in a certainproportion to each other; a sample obtained from it should include thesecell types in roughly similar proportions. However, when a sample ofcells obtained directly from a human specimen is extracted and expanded,one cell type may proliferate at a greater rate than the others, oranother cell type may not expand at all depending upon the conditionsemployed. Should that occur, the resulting sample would not berepresentative of the original specimen and thus may be compromised as aclinical sample.

A related requirement for preparation of a cell sample is that theresulting diseased cells retain their in vivo genomic and physiologicactivity profile so that they retain the physiological or genomiccharacteristics that will be tested. For instance, to measure celladhesion or cell signaling pathway activity, the cells' pathways ex vivomust retain their in vivo function. Otherwise, the accuracy of aclinical test result would be comprised. Another requirement is that anymethod used to prepare a cell sample must be capable of yieldingtestable cell samples from a high percentage of specimens obtained.

Current practices described in scientific literature for acquiringprimary cells from tissue for research report less than a 50% successrate in obtaining viable cell samples from tumor tissue (see e.g.,Crystal, A. S. et al. (2014) Science 346:1480-1486). The willingness ofclinicians to wait up to two weeks for a clinical test result requiresthe likelihood of a successful test to be high enough to justifydelaying treatment of their patient. Since delaying treatment for apatient entails risk, clinicians are unlikely to adopt a clinical testthat imposed this risk without a reasonable likelihood that a testresult of high utility will be available.

Thus, given the unique requirements of preparing a sample of viablediseased cells for clinical testing—preserving the viability of thecells during a transit period, availability of only a small amount ofspecimen, the short time available to expand the diseased cells,maintaining cell sample composition consistent with the original tumor,yielding a high percentage of testable cell samples, maintainingphysiological and genomic characteristics—there is a need for thedevelopment of novel methods of preparing primary cell samples derivedfrom human tissue specimens such that the sample is suitable for use inclinical testing.

SUMMARY OF THE INVENTION

The methods described herein address obstacles to preparing a sample ofviable diseased cells suitable for clinical testing. The inventionprovides compositions and methods of preparing a sample of viablediseased cells (i.e., primary cells) obtained from a subject byculturing said viable diseased cells in a media comprising anoikisinhibitors (e.g., intrinsic and/or extrinsic anoikis inhibitors), and/oron anti-anoikis, stress reducing surfaces (e.g., hydrated extracellularmatrix protein combinations), and/or in anti-anoikis, stress reducing,atmospheric conditions (e.g., greater than 2% and less than 20% oxygen)and especially during times of test sample preparation that are the mostlikely to cause the viable disease cells to enter into anoikis. Themethods of the invention allow for preparation of sufficient numbers ofprimary human cells, in a sufficiently short amount of time, thatmaintain the physiological and genomic characteristics of the cells invivo such that highly accurate clinical testing can be performed on themin a clinically relevant time frame.

Accordingly, in one aspect, the invention provides a method of preparinga sample of viable diseased cells obtained from a human subject forclinical testing, the method comprising:

culturing the sample of viable diseased cells obtained from the humansubject in a media comprising at least one anoikis inhibitor underconditions comprising greater than 2% and less than 20% oxygen; and

conducting a clinical test on the sample of viable diseased cells.

In another aspect, the invention provides a method of preparing a sampleof viable diseased cells obtained from a human subject for clinicaltesting, the method comprising:

culturing the sample of viable diseased cells obtained from the humansubject in a media comprising at least one anoikis inhibitor underconditions comprising 6-17% oxygen (e.g., 10% oxygen), e.g., for atleast one hour (e.g., for 3 hours);

culturing the sample in a media comprising at least one anoikisinhibitor and lacking digestion enzymes under conditions comprising6-17% oxygen (e.g., 10% oxygen), e.g., for at least one hour; (e.g., 3hours); and

culturing the sample in media lacking anoikis inhibitors underconditions comprising 20% oxygen, to thereby prepare the sample forclinical testing.

In another aspect, the invention provides a method of preparing a sampleof viable diseased cells obtained from a human subject for clinicaltesting, the method comprising:

culturing the sample of viable diseased cells obtained from the humansubject in a media comprising at least one anoikis inhibitor underconditions comprising 6-17% oxygen (e.g., 10% oxygen) on a cell culturevessel surface coated with a hydrated extracellular matrix (ECM).

In another aspect, the invention provides a method of preparing a sampleof viable diseased cells obtained from a human subject for clinicaltesting, the method comprising:

culturing the sample of viable diseased cells obtained from the humansubject in a media comprising at least one anoikis inhibitor underconditions comprising 6-17% oxygen (e.g., 10% oxygen) on a cell culturevessel surface coated with a hydrated extracellular matrix (ECM); and

attaching the sample of viable diseased cells to a surface comprising ahydrated extracellular matrix (ECM), wherein the ECM consists of: (i)fibronectin and collagen; (ii) collagen and laminin 332 (laminin V) or(iii) laminin 332 (laminin V), to thereby prepare the sample forclinical testing.

In yet another aspect, the invention provides a method of preparing asample of viable diseased cells obtained from a human subject forclinical testing, the method comprising:

culturing the sample of viable diseased cells obtained from the humansubject in a media comprising at least one caspase inhibitor, at leastone MMP3 inhibitor and at least one Rho-associated kinase inhibitorunder conditions comprising 6-17% oxygen (e.g, 10% oxygen), to therebyprepare the sample for clinical testing.

In yet another aspect, the invention provides a method of preparing asample of viable diseased cells obtained from a human subject forclinical testing using a biosensor, the method comprising:

attaching the sample of viable diseased cells to a surface comprising ahydrated extracellular matrix (ECM) consisting of fibronectin andcollagen; and

conducting a clinical test using a biosensor on the sample of viablediseased cells.

In yet another aspect, the invention pertains to a method of preparing asample of viable diseased cells obtained from a human subject forclinical testing comprising: culturing the sample of viable diseasedcells obtained from the human subject in a media comprising at least oneanoikis inhibitor (e.g., at least one inhibitor of an intrinsic anoikispathway and at least one inhibitor of an extrinsic anoikis pathway)under conditions comprising 6-17% oxygen (e.g., 10% oxygen);

attaching the sample of viable diseased cells to a surface comprising ahydrated extracellular matrix (ECM) consisting of fibronectin andcollagen; and

continuing culture of the sample attached to the surface in a mediacomprising at least one anoikis inhibitor under conditions comprising6-17% oxygen (e.g., 10% oxygen).

In one embodiment, the sample is cultured in a media comprising at leastone inhibitor of an intrinsic anoikis pathway and at least one inhibitorof an extrinsic anoikis pathway. In another embodiment, the sample iscultured in a media comprising at least one inhibitor of an intrinsicanoikis pathway and/or at least one inhibitor of an extrinsic anoikispathway inhibitor, or combinations thereof. In one embodiment, the atleast one apoptosis inhibitor agonizes an anti-anoikis pathway. In oneembodiment, the sample is cultured in a media comprising at least oneinhibitor of an anoikis pathway.

In one embodiment, the at least one anoikis inhibitor is selected fromthe group consisting of kinase inhibitors, protease inhibitors, stressinhibitors, death receptor inhibitors, cytochrome C inhibitors andanoikis inhibitors. In another embodiment, the at least one anoikisinhibitor is selected from the group consisting of Rho-associated kinaseinhibitors, ALK5 inhibitors, caspase inhibitors, matrix metalloproteaseinhibitors, redox buffering agents, reactive oxygen species inhibitors,TNFα inhibitors, TGFβ inhibitors, cytochrome C release inhibitors,carbonic anhydrase antagonists without calcium channel activation,integrin stabilizers, integrin ligands, Fas inhibitors, FasL inhibitors,Bax inhibitors and Apaf-1 inhibitors. Various non-limiting examples ofanoikis inhibitors are described further herein. In certain embodiments,the media in which the cell sample is cultured comprises two or moreanoikis inhibitors, such as two, three, four, five or more anoikisinhibitors. For example, in one embodiment, the sample is cultured in amedia comprising at least three anoikis inhibitors, such as aRho-associated kinase inhibitor, a caspase inhibitor and an MMP3inhibitor used in combination. Various other non-limiting examples ofcombinations of anoikis inhibitors are described further herein.

In one embodiment, the sample is cultured under conditions comprising6-17% oxygen. In another embodiment, the sample is cultured underconditions comprising 10% oxygen. In yet another embodiment, the sampleis cultured under conditions comprising 17-19% oxygen. In certainembodiments, the sample is cultured under two or more differentatmospheric conditions at different times, such as first culturing underconditions comprising 6-17% oxygen, such as at 10% oxygen for a periodof time, and then switching the sample to different atmosphericconditions, such as comprising 1-5% oxygen, or 17-19% oxygen, or 20%oxygen. Various suitable anti-anoikis, stress-reducing atmosphericconditions are described further herein.

In certain embodiments, the sample of viable diseased cells is contactedwith a digestion media for a period of time prior to culturing, whereinthe digestion media digests tissue without causing anoikis, cell surfaceadhesion molecule damage or non-anoikis means of cell death. Typically,the digestion media comprises one or more digestion enzymes. In certainembodiments, the digestion media also comprises at least one anoikisinhibitor and may contain multiple anoikis inhibitors (e.g., at leastone inhibitor of an intrinsic anoikis pathway and/or at least oneinhibitor of an extrinsic apoptotic pathway). In certain embodiments,the digestion media also comprises at least one anoikis inhibitor.

The method of the invention can comprise culturing the cell sample inmultiple different culture media for different periods of time and/orculturing the cell sample in culture media that comprise additionalcomponents in addition to the anoikis inhibitor(s). For example, in oneembodiment, the sample of viable diseased cells is cultured in a mediacomprising at least one component that promotes activation of adhesionpathways. Non-limiting examples of such components are described furtherherein. In another embodiment, the sample of viable diseased cells iscultured in a serum free culture media comprising at least one componentthat preserves physiological or genomic characteristics of the viablediseased cells. Non-limiting examples of such components are describedfurther herein. In another embodiment, the sample of viable diseasedcells is cultured in a serum free media comprising at least onecomponent that promotes cell growth, cell division or cell cycling tofacilitate cell proliferation. Non-limiting examples of such componentsare described further herein. In another embodiment, the sample ofviable diseased cells is cultured in a media comprising at least onecomponent that promotes temporary reversal of adhesion to facilitatetransfer of cells from one vessel to another vessel. Non-limitingexamples of such components are described further herein.

In certain embodiments, prior to conducting the clinical test, thesample of viable diseased cells is transferred to a media lackinganoikis inhibitors. In one embodiment, the sample of viable diseasedcells is cultured in the media comprising at least one anoikis inhibitorfor at least 120 hours before transfer to the media lacking anoikisinhibitors. In other embodiments, the sample is cultured in the mediacomprising at least one anoikis inhibitor for at least 24-96 hoursbefore transfer to the media lacking anoikis inhibitors. In oneembodiment, the sample of viable diseased cells is transferred to themedia lacking anoikis inhibitors under conditions comprising 20% oxygen.

In certain embodiments, prior to conducting the clinical test, thesample of viable diseased cells is attached to a surface comprising ahydrated extracellular matrix (ECM). In one embodiment, the hydrated ECMis folded. In one embodiment, the hydrated extracellular matrix (orhydrated and folded ECM) consists of fibronectin and collagen,preferably wherein the fibronectin and collagen comprise a fibrillic andhydrophilic surface. In another embodiment, the hydrated extracellularmatrix (or hydrated and folded ECM) consists of a collagen-laminin 332(laminin V) co-structure. In yet another embodiment, the hydratedextracellular matrix (or hydrated and folded ECM) consists of laminin332 (laminin V). In one embodiment, the surface is a biosensor surface.In another embodiment, the cells surface is a cell culture vesselsurface. The method can further comprise conducting a clinical testusing a biosensor on the sample of viable diseased cells.

The methods of the invention are suitable for use with a wide variety ofviable diseased cells obtainable from human subjects. In a preferredembodiment, the viable diseased cells are cancer cells. Furthermore, themethods of the invention are suitable for preparing primary human cellsfor a wide variety of in vitro clinical testing, including diagnostictests, genetic tests, treatment regimen tests and the like. In oneembodiment, the clinical test is conducted using a biosensor. In oneembodiment, the clinical test comprises contacting the sample of viablediseased cells with at least one agent and measuring cell adhesion orattachment before and after contact of the sample with the at least oneagent.

In another aspect, the invention pertains to a cell culture compositioncomprising primary human cancer cells cultured in a media comprising atleast one anoikis inhibitor under conditions comprising greater than 2%and less than 20% oxygen (e.g., 6-17% oxygen, 10% oxygen). In oneembodiment of the cell culture composition, the media comprises at leastone inhibitor of an intrinsic anoikis pathway and at least one inhibitorof an extrinsic anoikis pathway and wherein the cells are cultured underconditions comprising 6-17% oxygen (e.g., 10% oxygen). In anotherembodiment of the cell culture composition, the media comprises at leastthree anoikis inhibitors (e.g., a Rho-associated kinase inhibitor, acaspase inhibitor and an MMP3 inhibitor) and the cells are culturedunder conditions comprising 6-17% oxygen (e.g., 10% oxygen).

In another aspect, the invention pertains to a biosensor surfacecomprising primary human cancer cells attached to the biosensor surfacevia a hydrated extracellular matrix (ECM), wherein the ECM consists of:(i) fibronectin and collagen; (ii) collagen and laminin 332 (laminin V)or (iii) laminin 332 (laminin V). In one embodiment of the biosensorsurface, the ECM consists of fibronectin and collagen comprising afibrillic and hydrophilic surface. In one embodiment, the hydrated ECMis folded.

In another aspect, the invention pertains to a cell culture vesselsurface comprising primary human cancer cells attached to the cellculture vessel surface via a hydrated extracellular matrix (ECM),wherein the ECM consists of: (i) fibronectin and collagen; (ii) collagenand laminin 332 (laminin V) or (iii) laminin 332 (laminin V). In oneembodiment of the cell culture vessel surface, the ECM consists offibronectin and collagen comprising a fibrillic and hydrophilic surface.In one embodiment, the hydrated ECM is folded.

Kits for carrying out the methods of the invention, as well as methodsfor optimizing the cell culture conditions for culturing viable diseasecells from a human primary cell sample are also encompassed by theinvention.

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

DETAILED DESCRIPTION

The present invention describes methods of preparing samples of viablediseased cells extracted from tissue specimens obtained from humansubjects that are subsequently used while the cells are viable forclinical testing. These methods reduce the percentage of diseased (e.g.cancer) cells entering anoikis when human primary viable diseased cellsamples are extracted from tissue, cultured, and prepared for testing.Research on anoikis to date has largely been aimed at harnessing thecellular apoptosis system to kill cancer cells while preserving healthycell function. Anoikis research has not addressed the opposite functionof temporarily suspending anoikis in order to attain a stable testablestate of viable cultures of primary human cells, such as viable cancercells from a tumor sample.

In order to obtain and test viable cells, they must first be isolatedfrom tissue freshly removed from a patient. The present inventiondescribes methods to suspend the anoikis processes initiated once cellsare isolated from their native tissue so that it is possible to recoversufficient viable cells representative of their state in vivo. Thepresent invention also describes methods to prepare a cell sample withphysiological function and genomic composition comparable to thediseased cells when they were in vivo. The present invention alsodescribes methods to prepare a cell sample that attaches to a culturesurface in a manner that prevents the cell adhesion pathways frominterfering with cell signaling pathway function. The methods of theinvention involve the use of culture media comprising anoikis inhibitorsand/or the use of anti-anoikis, stress-reducing atmospheric conditionsand/or attachment of cells to anti-anoikis, stress reducing surfaces,and/or combinations thereof.

The utility and distinct advantages of the present invention areapparent by comparison of empirical results for different media with thepresent invention. Specifically, the present invention providesheterogeneous populations of cells capable of being cultured rapidlyfrom milligram amounts of disease tissue to provide uniform clinicaltest results. Morphological and biomarker analysis of cells culturedusing the compositions and methods of the present invention demonstratethe presence of multiple disease cell types (luminal, basal, stem,mesenchymal) replete with markers synonymous with disease (estrogenreceptor, progesterone receptor, ErbB family receptors, CD10, claudin 4,CD49f, and other markers described herein) whereas other media known andcommercially available in the art are incapable of generating suitablesamples of diseased cells for clinical testing. Other media types do notsupport the propagation of luminal diseased cell types that alsotypically characterize diseased cell patient samples. Furthermore, othermedia types support only healthy cells or stem cells or squamous celltypes that are not in keeping with the desired outcome of the presentinvention for a heterogeneous mixture of diseased cells from a patientsample. In summary the present invention provides cells of heterogeneoustypes with identified markers from a sample of primary diseased cellsfrom a subject suitable for clinical testing whereas other media knownin the art do not.

Various aspects of the invention are described further in thesubsections below.

Inhibition of Anoikis

An obstacle to the successful culturing of primary cells, such as asample of viable diseased cells from a subject such that the cells canbe used in clinical testing, is anoikis, part of the natural controlsystem in cells to program death. Programmed death may be initiated inresponse to many factors, potentially acting together, including, butnot limited to, buildup of intolerable numbers of mutations, pH, cellage, environmental factors, various forms of stress such as high or lowextremes of oxygen partial pressure, contact with cytokines, chemokines,cell signaling molecules, proteins, and various RNA, and cell growthbeyond established tissue boundaries. Balance of programmed cell deathactivity is attained in cells by interlinked protein pathways that areeither pro-anoikis or pro-angiogenic. For example, multi-domainpro-anoikis family members such as BAX and BAK, once activated,permeabilize mitochondria to trigger anoikis, whereas anti-anoikis BCL-2family members preserve mitochondrial integrity. The BH3-only molecules(BH3s) promote anoikis by either activating BAX-BAK or inactivatinganti-anoikis members.

Two forms of anoikis pathways have been described, intrinsic andextrinsic (see e.g., Fulda, S. and Debatin, K. M. (2006) Oncogene7:4798-4811. The intrinsic anoikis pathway is characterized bymitochondrial membrane permeabilization, cytochrome c release, andapoptosome formation in response to cytotoxic stimuli. Extrinsic anoikispathways are activated in response to ligand binding to death receptorsof the TNF-superfamily. Exemplary ligands include TNFα and FasL. Deathreceptor activation can result in mitochondrial membranepermeabilizaton, cytochrome c release, and through caspase dependentcleavage and translocation of the pro-anoikis protein Bid tomitochondria, progress is made to later stages of anoikis. Bothintrinsic and extrinsic pathways involve mitochondrial membranepermeabilization, cytochrome C release, and caspase activation as sharedcomponents.

Inhibition of anoikis in the present invention focuses on prevention andaddressing the early, initiating, anoikis events for the sake ofinhibition efficiency. Interruption of anoikis processes can beperformed at later points before cell death and may also be desirable torevive cell culture samples that have already entered late stages ofanoikis. Buffering of pH and redox potential and metal ion concentration(especially release of calcium) of the diseased primary cells arepreferred early points of preventing anoikis. Early points of inhibitionof anoikis preferably include disruption of mitochondrial permeabilityand inhibition of cytochrome c release. Somewhat later points ofintervention in anoikis processes preferably include disruption of thecaspase activity cascade and disruption of other proteases. Deregulatedanoikis with increased expression of anti-anoikis molecules of the Bcl-2family or reduction of pro-anoikis molecules such as Bax, Apaf-1 orcaspase-8 are additional embodiments that may be combined to inhibitanoikis.

Accordingly, in the methods of the invention, a sample of viablediseased cells obtained from a subject is cultured in a media comprisingat least one anoikis inhibitor. In certain embodiments, the culturemedia comprises at least one intrinsic anoikis inhibitor and at leastone extrinsic anoikis inhibitor. In other embodiments, the mediacomprises at least one intrinsic anoikis inhibitor and at least oneextrinsic anoikis inhibitor. In other embodiments, the apoptosisinhibitors are selected to agonize anti-anoikis pathways.

Following any initial preparatory steps on the tissue specimen to obtaincells suitable for culture (discussed further below), the cellsextracted from the specimen are cultured in a media comprising anoikisinhibitors and more preferably a combination of anoikis inhibitors, mostpreferably combinations that are selected from intrinsic and orextrinsic pro-anoikis pathway inhibitors and or anti-anoikis activators.

Table 1 below shows non-limiting examples of general types of anoikisinhibitors and exemplary classes of inhibitors that fall within thosetypes, as well as examples of specific reagents and whether theinhibitor affects the intrinsic anoikis pathway, the extrinsic anoikispathway or a component common to both pathways (referred to herein as“shared pathway” anoikis inhibitors).

TABLE 1 Exemplary Anoikis Inhibitor Molecules General Inhibitor Specificreagent Type Exemplary Classes examples Pathway Kinase inhibitorRho-kinase Y-27632 Shared inhibitor Ceritinib, RepSox Extrinsic ALK5inhibitor Protease inhibitor Caspase inhibitor Z-VAD-FMK Shared MatrixMetallo- UK-356618 Shared Protease inhibitor Stress inhibitor Redoxbuffering Niacin Intrinsic Reactive Oxygen Glutathione ethyl IntrinsicSpecies inhibitor ester Death Receptor TNF alpha Infliximab Extrinsicinhibitor TGF beta Fresolumimab Extrinsic Cytochrome C Release inhibitorMelatonin Shared inhibitor Carbonic Methazolamide Shared anhydraseantagonist without Ca²⁺ channel activation Adhesion-related Integrinstabilizer Tetraiodothyronine Extrinsic anoikis inhibitor Integrinligand Fibrinogen, Extrinsic fibronectin

In one embodiment, the at least one anoikis inhibitor is aRho-associated kinase inhibitor, non-limiting examples of which includeY-27632, GSK429286A and RKI-1447. In one embodiment, the at least oneanoikis inhibitor is a caspase inhibitor, such as a pan caspaseinhibitor (e.g., Z-VAD-FMK), a caspase 3 inhibitor (e.g., Z-DEVD-FMK,Q-VD-OPh), a caspase 8 inhibitor (e.g., Z-IETD-FMK, Ac-LETD-CHO,Q-VD-OPh) and/or a caspase 9 inhibitor (e.g., Z-LEHD-CHO, Ac-LETD-CHO,Q-VD-OPh). In one embodiment, the at least one anoikis inhibitor is acytochrome C inhibitor, non-limiting examples of which include melatoninand methazolamide. In one embodiment, the at least one anoikis inhibitoris a Matrix Metalloprotease (MMP3) inhibitor, a non-limiting example ofwhich is UK-356618. In one embodiment, the at least one anoikisinhibitor is an ALK5 inhibitor, non-limiting examples of which includeceritinib, RepSox, alectinib, AP26113, GW788388, SD-208 and Galunisertib(LY2157299). In one embodiment, the at least one anoikis inhibitor is aTNFα inhibitor, non-limiting examples of which include R-7050,infliximab, golimumab, adalimumab, certolizumab and etanercept. In oneembodiment, the at least one anoikis inhibitor is a TGFβ inhibitor,non-limiting examples of which include fresolimumab, as well asinhibitors of TGFβ pathway signaling, such as ceritinib, RepSox,alectinib, AP26113, GW788388, SD-208 and Galunisertib (LY2157299). Inone embodiment, the at least one anoikis inhibitor is an oxidativestress reducer (i.e., a Reactive Oxygen Species inhibitor), non-limitingexamples of which include glutathione, glutathione ethyl ester,methionine, cystine, cysteine, glutamic acid and glycine. In oneembodiment, the at least one anoikis inhibitor is a redox bufferingagent, non-limiting examples of which include niacin, niacin-relatedcompounds, calciferols, beta carotene and vitamin C. In one embodiment,the at least one anoikis inhibitor is an adhesion-related anoikisinhibitor, non-limiting examples of which include fibrinogen,fibronectin, and tetraiodothyronine. In one embodiment, the at least oneanoikis inhibitor is a Fas and/or FasL inhibitor, non-limiting examplesof which include triiodothyronine and tetraiodothyronine. In oneembodiment, the at least one anoikis inhibitor is a Bax inhibitor,non-limiting examples of which include Bax inhibitor-1 and V5 peptide.In one embodiment, the at least one anoikis inhibitor is an Apaf-1inhibitor, non-limiting examples of which include QM31 and minocycline.

In other embodiments, cells are cultured with two or more anoikisinhibitors, such as with two, three, four, five, six, seven, eight, nineor ten anoikis inhibitors. Any of the aforementioned anoikis inhibitorscan be used in combination. For example, in one embodiment, the cellsare cultured with a Rho-associated kinase inhibitor and a caspaseinhibitor. In another embodiment, the cells are cultured with aRho-associated kinase inhibitor and an MMP3 inhibitor. In anotherembodiment, the cells are cultured with a caspase inhibitor and an MMP3inhibitor. In another embodiment, the cells are cultured with aRho-associated kinase inhibitor, a caspase inhibitor and an MMP3inhibitor. In another embodiment, the cells are cultured with aRho-associated kinase inhibitor, a caspase inhibitor, an MMP3 inhibitorand a Death Receptor inhibitor (e.g., a TNFα inhibitor, a TNFR1inhibitor, a TGFβ inhibitor, an ALK5/TGFBR1 inhibitor, a Fas inhibitoror a FasL inhibitor). In another embodiment, the cells are cultured witheach of: a kinase inhibitor, a protease inhibitor, a stress inhibitor, aDeath Receptor inhibitor, a cytochrome C inhibitor and an anoikisinhibitor, non-limiting examples of which include the reagents set forthin Table 1 and listed above. Other suitable combinations of anoikisinhibitors can be determined using the guidance provided herein (e.g.,see Examples 3-5).

Inhibitors typically are added to the culture media at a concentrationof 0.001-0.1 micromole per 100,000 cells, preferably at 0.01-0.1micromole per 100,000-10,000,000 cells, depending upon the amount ofcells in a diseased cell sample.

In certain embodiments, use of a single anoikis inhibitor in the culturemedia provides benefit to certain cell types. However, in specificembodiments, it is the benefit of combining the intrinsic, extrinsic,and anoikis inhibition components that provides the most optimalconditions for preparing the sample for clinical testing. It has beenobserved that this combination of anti-anoikis components renders aheterogeneous population of diseased cells from a significant majorityof specimens from different patients suitable for clinical testing ofviable diseased cells. Each patient specimen may enter anoikis for anyof a number of different individual or combined reasons describedpreviously, each with the ability to rapidly and catastrophically makethe specimen un-useful for any kind of viable cell test. Only throughthe significant attenuation of a combination of these routes to anoikiscan the sample of heterogeneous diseased cells be made stable uponremoval from tissue for viable cell testing. Clinical testing requiresthat most samples that are received for testing reliably provide aphysician with test result information to guide the treatment of theirpatients. The present invention provides the means of accomplishing thisrequirement.

Anti-Anoikis Atmospheric Conditions

Another factor affecting whether a primary cell cultured in vitro entersanoikis has been found to be the atmospheric conditions under which thecells are cultured. It has now been discovered that combining the use ofat least one anoikis inhibitor in the media with culture underconditions of greater than 2% and less than 20% oxygen serves to furtherinhibit anoikis of primary cells in the sample of viable diseased cells.While not being bound by mechanism, it is thought that culturing thecells in atmospheric conditions of greater than 2% and less than 20%oxygen reduces anoikis associated with oxidative stress. In anotherembodiment, the cell sample is maintained in atmospheric conditions thatpermit glycolysis and mitochondrial function conducive to cellproliferation. In a more preferred embodiment, the cell culture ismaintained in atmospheric conditions that balance the reduction inanoikis associated with oxidative stress and the requirement for oxygenconducive to cell proliferation. A most preferred embodiment is tomaintain the cell sample in atmospheric conditions comprising 6%-17%oxygen, a range variant that is between the hypoxic conditions the cellsexperience in vivo (1-5% oxygen) and the normoxic conditions the cellsexperience when exposed to the atmosphere (20% oxygen). In anotherembodiment, the cells are cultured under conditions comprising 10%oxygen. In yet other embodiments, the cells are cultured underconditions comprising 6-9% oxygen, 9-11% oxygen, 8-12% oxygen, 7-13%oxygen, 6-14% oxygen, 11-13% oxygen, 13-15% oxygen, 15-17%, 9-15%oxygen, 6% oxygen, 7% oxygen, 8% oxygen, 9% oxygen, 11% oxygen, 12%oxygen, 13% oxygen, 14% oxygen, 15% oxygen, 16% oxygen or 17% oxygen. Inanother embodiment, cells cultured under conditions comprising 1-5%oxygen.

In certain embodiments, cells are cultured under two or more differentatmospheric conditions for different periods of time. For example, thecells can first be cultured under conditions comprising 6-17% oxygen(e.g., 10% oxygen) and then cultured under conditions comprising 1-5%oxygen, or comprising 17-19% oxygen or comprising 18-20% oxygen orcomprising 20% oxygen.

Other aspects of the atmospheric conditions and temperature for cellculture are those typically used for culture of mammalian (e.g., humancells). For example, the cells typically are cultured in atmosphericconditions comprising at least 5% carbon dioxide, relative humidity (RH)between 40%-100% (e.g., 85% RH) and at a temperature of 37° C.

Surface Attachment and Inhibition of Anoikis

Most non-transformed and many cancer epithelial cell types undergoanoikis when they lose their contact with the hydrated extracellularmatrix (ECM), a phenomenon termed adhesion-related anoikis. Maintenanceof the structural and functional integrity of the epithelial cellrequires highly dynamic cell-cell and cell-matrix interactions involvingdifferent types of surface receptors. Among these receptors are adhesionmolecules, such as cadherins and integrins, which play a major role byrecognizing and interacting with other cell adhesion receptors onneighboring cells and by binding components of the ECM. Besidesproviding mechanical anchorage to the cell, these structures also are offunctional importance; they transduce signals from the ECM andneighboring cells that are critical for survival and proliferation. Thecomposition, patterning, and structural nature of the ECM has beendemonstrated to be critical in restoring and or establishing theviability of mammalian cells. Reduction or loss of these contacts orsignals or their integrity frequently initiates anoikis.

Reduction of anoikis for epithelial cells is critical to preparinguniform samples of viable diseased cells, especially for clinical testpurposes. Adhesion-related anoikis is a form of anoikis that isinitiated in vivo by anchorage-dependent cells such as epithelial cells,becoming detached from the surrounding tissue extracellular matrix(ECM). Usually cells stay close to the tissue to which they belong sincethe communication between proximal cells as well as between cells andECM provides essential signals for growth or survival. When cells aredetached from the ECM, there is a loss of normal cell-matrixinteractions, which leads to forms of intrinsic and extrinsic anoikis.By their very nature, diseased cells often have heterogeneous attachmentstability. In cancer, poor attachment is characterized pathologically asmetastasis, a highly fatal form of the disease. Preparation of uniformsamples of diseased cells for clinical testing necessary involves theirremoval from their ECM in their native environment, which therefore caninduce anoikis.

Therefore, various embodiments of the methods of the present inventionreplace the ECM from which the cells were removed with an in vitro ECMthat restores cell-cell and cell-ECM contacts, thereby inhibiting orreducing anoikis. Furthermore, one or more components can be added tothe culture media to promote restoration and/or activation of cell-celland or cell-ECM adhesion and to prevent anoikis.

Accordingly, certain embodiments of the methods of the invention includeuse of a culture media comprising at least one component that promotesrestoration and or activation of cell-cell and or cell-ECM adhesion andprevents anoikis. The adhesion pathway activators that prevent this formof anoikis activity can be selected from a group consisting of but notlimited to Ca²⁺, Mg²⁺, Mn²⁺, triiodothyronine, tetraiodothyronine,fetuin, solution forms (e.g. fibrinogen) or coatings of extracellularmatrix components or combinations of such components such asfibronectin, collagens, laminins, vitronectin, intercellularCAMs,VascularCAMS, MAdCAMS, glycosaminoglycans, proteoglycans or derivativesand or peptides thereof. Growth factors such as EGF to increase focaladhesion kinase activity and counteract PTEN anoikis promotion followingintegrin disruption are also included in the present invention.

In certain embodiments, two or more of the above components that inhibitanoikis can be used in the culture media. For example, in oneembodiment, the sample of viable diseased cells is cultured in a mediacomprising at least one intrinsic anoikis inhibitor and at least oneextrinsic anoikis inhibitor.

In further embodiments, the viable cell sample is attached to a culturesurface, such as a culture vessel (e.g., culture plates divided up intowells, T25, T75, and T150 flasks, multilevel culture vessels and thelike) or a test surface (e.g., a biosensor surface) via an extracellularmatrix. In other embodiments, the viable cell sample is attached to acell culture vessel or test surface via a hydrated ECM where the ECMretains sufficient water such that the ECM is fully wetted and is neverallowed to dehydrate prior to use. In additional embodiments, the viablecell sample is attached to a cell culture vessel or test surface via ahydrated and folded ECM whereby the ECM is comprised of an ordered,structured, and reproducible arrangement of its protein component(s). Inembodiments, the cell sample of viable cells is attached to a fibrillicand hydrophilic combined collagen and fibronectin surface prepared tomaintain these proteins' natural in vivo-like structures that can berecognized by specific integrins and or promote cell-cell interactionsvia for example cadherins or adherins. In certain embodiments, thecollagen and fibronectin are formed into hydrophilic fibrils and thetwo-protein interaction is comprised of specific amino acid sequencebinding sites. In embodiments, the cell-ECM attachment forms acollagen-laminin 332 co-structure. Laminin 332 is also known as lamininV and in certain embodiments can be used as a single ECM on the culturesurface. In some embodiments, the surface to be coated with ECM isplastic, glass, or especially glass imprinted with gold electrodes, ortitanium coated plastic, or optically active metal coated glass orplastic formed into a wave-guide, gold coated glass. Other preferredembodiments the surface to be coated with ECM is a biosensor forclinical test purposes.

In each of the embodiments involving surface attachment, the surfacecoating is prepared using standard methods known to those skilled in theart. For example, methods of preparing a hydrated ECM or a hydrated andfolded ECM are well established in the art (see e.g., Anderson, D. G. etal. (2004) Nat. Biotechnol. 22:863-866; Flaim, C. J. et al. (2005) Nat.Methods 2:119-125; Falsey, J. R. et al. (2001) Bioconjug. Chem.12:346-353; Kuschel, C. et al. (2006) Biotechniques 40:523-531;Reticker-Flynn, N. E. et al. (2012) Nat. Commun. 3:1122).

In other embodiments, the cell attachment to a surface is optimized toensure that the level of cell attachment is not driven by conditionsthat stress the cells or conditions that increase anoikis, which mayresult in cell attachment that interferes with cell signaling activity.In other embodiments, the cell attachment to the surface is optimized toensure that extracellular coating and adhesion mechanism are matched tothe signaling pathway of interest.

Accordingly, in certain embodiments, the methods of the invention forpreparing primary cells for conducting a clinical test comprisesinclusion of step comprising attaching the sample of cells to a surfacecomprising a hydrated extracellular matrix (ECM). In one embodiment, theECM consists of fibronectin and collagen. In one embodiment, folded ECMis used. In another embodiment, fibronectin and collagen comprise afibrillic and hydrophilic surface. In one embodiment, the ECM consistsof a fibrillic and hydrophilic collagen-laminin 332 (laminin V)co-structure. In another embodiment, the ECM consists of laminin 332(laminin V). The invention provides a method of preparing primary cellsfor conducting a clinical test, the method comprising:

attaching the sample of viable diseased cells to a biosensor surfacecomprising a hydrated extracellular matrix (ECM), or a hydrated andfolded ECM, wherein the ECM consists of: (i) fibronectin and collagen;(ii) collagen and laminin 332 (laminin V) or (iii) laminin 332 (lamininV); and

conducting a clinical test using a biosensor on the sample of viablediseased cells.

In certain embodiments, the sample of viable diseased cells may beattached to two or more different surfaces at different time periods.For example, the cells initially may be attached to a cell surface suchas a culture plate, culture flask or other culture vessel comprising ananti-anoikis, stress-reducing ECM as described above and cultured for aperiod of time. Then, the cells may be detached from this surface (e.g.,as described further below) and subsequently attached to anothersurface, such as a biosensor surface for clinical testing. Thisbiosensor surface also can comprise an anti-anoikis, stress-reducing ECMas described above.

Use of Multiple Culturing Conditions in Combination

Regulation of in vivo cell viability and function is known to be highlycomplex, relying upon multiple signal inputs and responses to maintainstable cellular processes. This is especially true for the effect thecell microenvironment has on cells. Culturing human primary cells in thelaboratory setting has been characterized by failure and a prevailingmindset that highly reproducible primary tissue culturing is notpossible, especially of diseased cells. This mindset has also blockedthe development of any live primary cell functional clinical test todate. Weighing heavily on this history of failure has been the lack ofconsideration of the need to address the multiple signal inputs andresponses required by primary cells to establish a viable culture exvivo. When different diseased cells are cultured ex vivo, they willundergo anoikis as their natural regulatory control signaling isdisrupted. In order to overcome this regulatory control for thepreparation of diseased cells for clinical testing, a combination ofmethods is required to address the regulatory signal maintenancerequirement and prevent anoikis. The methods of the invention forpreparing primary cells encompass the use of multiple different culturemedia and/or culture conditions that are used in combination to preparethe primary cells for clinical testing. The use of different culturemedia and/or culture conditions in combination further improves thepreparation of the primary cells such that sufficient numbers of cellsare obtained in a sufficient period of time, while maintaining thephysiological and genomic characteristics of the cells, such that theyprovide reliable results in clinical testing.

In certain embodiments, the cell sample is cultured first in a mediacomprising anoikis inhibitor(s) and then subsequently cultured in amedia that does not include anoikis inhibitor(s). In certainembodiments, the cell sample is maintained first in an atmospherecomprising greater than 2% and less than 20% oxygen (e.g., greater than3% and less than 19% oxygen, 6-17% oxygen, 10% oxygen, or any of theother atmospheric conditions described herein above 2% and below 20%)and subsequently maintained in an atmosphere comprising 20% oxygen. Incertain embodiments, the period of time the cell sample is cultured in amedia comprising anoikis inhibitor(s) ranges from 1-10 hours, from 10-20hours, from 20-30 hours, from 30-40 hours, from 40-50 hours, or morethan 50 hours. For example, the cell sample can be cultured in a mediacomprising anoikis inhibitor(s) for at least 12 hours, at least 24hours, at least 48 hours, at least 96 hours, at least 120 hours, 24-48hours, 24-96 hours, or 24-120 hours. In certain embodiments, the periodof time the cell sample is maintained in an atmosphere with greater than2% and less than 20% oxygen ranges from 1-10 hours, from 10-20 hours,from 20-30 hours, from 30-40 hours, from 40-50 hours, or more than 50hours. For example, the cell sample can be maintained in an atmospherewith greater than 2% and less than 20% oxygen for at least 12 hours, atleast 24 hours, at least 48 hours, at least 96 hours, at least 120hours, 24-48 hours, 24-96 hours, or 24-120 hours.

In other embodiments, the cell sample is maintained in an atmospherecomprising greater than 2% and less than 20% oxygen for a period of timeafter the cells are transferred from one culture vessel to anothervessel. In certain embodiments, the period of time the cell sample ismaintained in an atmosphere comprising greater than 2% and less than 20%oxygen after the cells are transferred from one culture vessel toanother vessel ranges from 1-10 hours, from 10-20 hours, from 20-30hours, from 30-40 hours, from 40-50 hours, or more than 50 hours. Apreferred embodiment is to culture the cell sample in atmosphericconditions comprising 6% -17% oxygen after the cells are transferredfrom one culture vessel to another vessel. In certain embodiments, theatmospheric conditions maintained after the cell sample is transferredto a new culture vessel comprise oxygen that ranges between 1-5%, 6-9%,9-11%, 8-12%, 7-13%, 6-14%, 11-14%, 14-17%, or 17%-20% or conditions of6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16% or 17% oxygen. Inembodiments, the cell sample is maintained first in an atmospherecomprising greater than 2% and less than 20% oxygen after the cells aretransferred from one culture vessel to another culture vessel andsubsequently placed in an atmosphere comprising 20% oxygen.

In other embodiments, the cell sample is cultured in a media comprisinganoikis inhibitor(s) for a period of time after the cells aretransferred from one culture vessel to another vessel. In certainembodiments, the period of time the cell sample is cultured in a mediacomprising anoikis inhibitors after the cells are transferred from oneculture vessel to another vessel ranges from 1-10 hours, from 10-20hours, from 20-30 hours, from 30-40 hours, from 40-50 hours, or morethan 50 hours. In embodiments, the cell sample is cultured first in amedia comprising anoikis inhibitor(s) after the cells are transferredfrom one culture vessel to another culture vessel and subsequentlyplaced in a culture media that does comprise anoikis inhibitor(s).

In certain embodiments, cells are cultured in a base media supplementedwith various components. In one embodiment, the base media is a serumfree base media. In another embodiment, the base media comprises anoikisinhibitor(s) as described herein and may contain additional componentsserving additional functions as described below. Non-limiting examplesof base media that are serum-free that could be modified by addition ofcomponents described herein include DMEM, F12, 50% DMEM/50% F12, MEGM,MCDB-170.

In other embodiments, the cells are cultured in serum free culture mediacomprising at least one component that promotes growth, division, orcell cycling to facilitate cell proliferation. The media containing thistype of component can be the same media that contains the anoikisinhibitor(s) or can be a different media into which the cells aretransferred. Non-limiting examples of such components that promotegrowth, division, or cell cycling to facilitate cell proliferationinclude growth factors associated with the tumor microenvironment, HGF,FGF (types 1-4), exosomes, miRNAs, other small non-protein coding RNAs,longer non-protein coding RNAs, hormones, IGF, VEGF, EGF, interleukins,cytokines, chemokines, and other factors such as may be known asautocrine or paracrine factors produced by cells in and around thediseased cells.

In other embodiments, the culture media is comprised of at least onecomponent that promotes temporary reversal of adhesion activity tofacilitate gentle transfer of a cell culture from one vessel to anothervessel. The media containing this type of component can be the samemedia that contains the anoikis inhibitor(s) or can be a different mediainto which the cells are transferred. Non-limiting examples of suchcomponents that promote temporary reversal of adhesion activity tofacilitate gentle transfer of a cell culture from one vessel to anothervessel include non-enzymatic and enzymatic treatments, Ca²⁺, Mg²⁺, andor Mn²⁺ chelators such as EGTA, EDTA, and other divalent metal ionchelators known to those practiced in the art, and additionally dispase,TrypLE, trypsin, accutase, accumax, collagenase, hyalronidase, elastase,trypsin inhibitor, STEMxyme, pronase, deoxyribonuclease. Combinations ofthe members of this group at differential concentrations, applied fordifferent amounts of time at different temperatures are included in thepresent embodiment.

In other embodiments, the culture media is a serum-free culture basemedia comprising at least one component that preserves the physiologicalor genomic characteristics of the diseased cells. The media containingthis type of component can be the same media that contains the anoikisinhibitor(s) or can be a different media into which the cells aretransferred. Non-limiting examples of such components that preserve thephysiological or genomic characteristics of the diseased cells includeessential and non-essential amino acids, vitamins, especially Bvitamins, rare essential metals, pH buffer(s) (e.g. N-[2-HydroxyEthyl]Piperazine-N′-[2-EthaneSulfonic acid] also known as HEPES, NaHPO₄,NaH₂PO₄), salts (Na+, K+, Fe, Zn, Cu, Mg²⁺, Ca²⁺, Mn²⁺, Cl, CO₃, SO₄),selenites, silicates, glucose or sugars or other biologically practicalcarbon sources, lipids, fatty acids such as lipoic acid, pyruvate, BSA(e.g also includes reagents such as Albumax or other serum albumins),transferrin, growth factors, chemokines, cytokines, mitogens,ethanolamine, phosphoethanolamine, albumins, hormones (e.g.progesterone, testosterone, estradiol—especially 17β-estradiol,hydrocortisone, insulin), putrescine, pyruvate, thymidine, linoleicacid, folic acid, folinic acid, choline, pyridoxal hydrochloride,biotin, hypoxanthine, purine and purine derivatives, pyrimidines andpyrimidine derivatives, antibiotics, anti-fungals, anti-mycotics. Themedia components can be selected to support diseased epithelial cellgrowth and said selected components can cause separation from the growthof healthy, disease-free, epithelial cells. Growth factors, cytokines,and chemokines are added at 0.001-0.1 nmol per 100,000 cells, preferablyat 0.01-0.1 nmol per 100,000-1,000,000 cells, depending upon the amountof cells in a diseased cell sample.

In one embodiment of a combination method of the invention for preparinga sample of viable diseased cells obtained from a human subject forclinical testing, the method comprises:

culturing the sample of viable diseased cells obtained from the humansubject in a media comprising at least one inhibitor of an intrinsicanoikis pathway and at least one inhibitor of an extrinsic anoikispathway under conditions comprising 6-17% oxygen (e.g., 10% oxygen); and

conducting a clinical test on the sample of viable diseased cells.

In another embodiment of a combination method of the invention forpreparing a sample of viable diseased cells obtained from a humansubject for clinical testing, the method comprises:

culturing the sample of viable diseased cells obtained from the humansubject in a media comprising at least one digestion enzyme and at leastone anoikis inhibitor under conditions comprising 6-17% oxygen (e.g.,10% oxygen), for example for at least one hour (e.g., 1-3 hours, 3 hoursor no more than 3 hours);

culturing the sample in a media comprising at least one anoikisinhibitor and lacking digestion enzymes under conditions comprising6-17% oxygen (e.g., 10% oxygen), for example for at least 10 hours, orat least 24 hours, or at least 48 hours or least for 96 hours, or atleast 120 hours, or for 24-48 hours, or for 24-96 hours or for 24-120hours; and

culturing the sample in media lacking anoikis inhibitors underconditions comprising 20% oxygen, to thereby prepare the sample forclinical testing.

In another embodiment of a combination method of the invention forpreparing a sample of viable diseased cells obtained from a humansubject for clinical testing, the method comprises:

culturing the sample of viable diseased cells obtained from the humansubject in a media comprising at least one inhibitor of an intrinsicanoikis pathway and at least one inhibitor of an extrinsic anoikispathway under conditions comprising 6-17% oxygen (e.g., 10% oxygen); and

attaching the sample of viable diseased cells to a surface comprising ahydrated extracellular matrix (ECM), wherein the ECM consists of: (i)fibronectin and collagen; (ii) collagen and laminin 332 (laminin V) or(iii) laminin 332 (laminin V).

In one embodiment, the surface to which the sample of viable diseasedcells is attached is a biosensor surface, the method further comprisingconducting a clinical test using a biosensor on the sample of viablediseased cells.

In another embodiment of a combination method of the invention forpreparing a sample of viable diseased cells obtained from a humansubject for clinical testing, the method comprises:

culturing the sample of viable diseased cells obtained from the humansubject in a media comprising at least one anoikis inhibitor underconditions comprising 6-17% oxygen (e.g., 10% oxygen);

attaching the sample of viable diseased cells to a surface comprising ahydrated extracellular matrix (ECM) consisting of fibronectin andcollagen; and

continuing culture of the sample attached to the surface in a mediacomprising at least one anoikis inhibitor under conditions comprising6-17% oxygen (e.g., 10% oxygen). In one embodiment, the sample iscultured with at least one inhibitor of an intrinsic anoikis pathway andat least one inhibitor of an extrinsic anoikis pathway. In oneembodiment, the surface to which the sample of viable diseased cells isattached is a biosensor surface, the method further comprisingconducting a clinical test using a biosensor on the sample of viablediseased cells.

In yet another embodiment of a combination method of the invention forpreparing a sample of viable diseased cells obtained from a humansubject for clinical testing, the method comprises:

culturing the sample of viable diseased cells obtained from the humansubject in a media comprising at least one digestion enzyme (e.g.,collagenases and hyaluronidase) and at least one anoikis inhibitor(e.g., an intrinsic anoikis inhibitor(s) and/or an extrinsic anoikisinhibitor(s)) under conditions comprising 6-17% oxygen (e.g., 10%oxygen) for no more than three hours;

plating the sample of viable diseased cells onto a cell culture vesselsurface comprising a hydrated and folded extracellular matrix (ECM)consisting of human collagen I and human fibronectin and culturing in amedia comprising at least one anoikis inhibitor (e.g., an intrinsicanoikis inhibitor(s) and/or an extrinsic anoikis inhibitor(s)) underconditions comprising 6-17% oxygen (e.g., 10% oxygen); and

culturing the sample in a serum free base media until a clinical test onthe viable disease cells is performed or until 50%-80% confluency of theviable disease cells is attained.

This combination method can include additional steps after the lastculturing steps. For example, in one embodiment, the primary cells areharvested by culture of the cells in a media comprising at least onecomponent that promotes temporary reversal of adhesion activity (e.g.,minimal enzyme and divalent metal ion conjugating media) for no morethan 10 minutes, under conditions that reverse attachment of the viabledisease cells to the surface without damaging the adhesion components ofthe cell. In another embodiment, the combination method can furthercomprise transfer of the harvested cells to a surface (e.g., a secondculture vessel, or a biosensor surface) comprising a hydrated and foldedextracellular matrix (ECM) (e.g., consisting of human collagen I andhuman fibronectin) and culture in a media comprising at least oneanoikis inhibitor under conditions comprising 6-17% oxygen (e.g., 10%oxygen) for at least 1-8 hours, e.g., until significant attachment andspreading is observed.

Following culture of primary cells according to the methods of theinvention for preparing the cells for clinical testing, the methods canalso include identification and characterization of diseased cellpopulations. In one embodiment, diseased cell populations are identifiedand/or characterized by detection of specific markers, such as thoselisted in Table 2, e.g, by FACS or by qPCR methods. Additionally, oralternatively, diseased cell populations can be identified and/orcharacterized by testing of adhesion and/or cell signaling pathwayactivity, e.g., using a biosensor.

Cell Samples and Initial Preparation Thereof

The methods of the invention can be applied to a wide variety of primarycells, in particular primary human cells, in particular viable diseasedcells from a human subject. In on embodiment, the cells are cancer cellsfrom a tumor sample.

In embodiments, the cell sample is extracted from a fresh solid cancertissue specimen obtained from a core needle biopsy, fine needleaspiration, vacuum-assisted core biopsy, image-guided core needlebiopsy, surgical biopsy, surgical resection, or any other suitablemedical procedure.

In embodiments, the cell sample is obtained from any biological tissueor fluid type, either diseased or normal.

In preferred embodiments, diseased epithelial cells may be derived frombreast tissue, stomach tissue, colon/intestinal tissue, lung tissue,head tissue, neck tissue, parotid tissue, ovarian tissue, uterinetissue, cervical tissue, prostate tissue, pancreatic tissue,kidney/renal tissue, or bone and/or bone marrow. In specificembodiments, the tissue is derived from patients with the canonicalbreast cancer pathological types—for example those described by clinicalpathological markers: ER+/HER2−, ER+/HER2+, ER−/HER2+, ER−/HER2.

In embodiments, culture media comprised of different components ordifferent concentrations of components are used at different stages ofcell preparation. In embodiments, the cell sample is prepared using one,two, three, four or more than four different culture media. Inembodiments, the cell sample is maintained in atmospheric conditionscomprised of different concentrations of oxygen at different stages ofcell preparation. In embodiments, one, two, three, four or more thanfour different atmospheric conditions are used at different stages ofcell preparation. In other embodiments, different culture media, andanoikis inhibitors, and atmospheric conditions, and attachmentconditions are used together in any combination.

To prepare a diseased cell sample, a tissue specimen typically is firstmechanically divided into smaller pieces and placed in a digestion mediacontaining enzymatic agents known to digest tissue (i.e., digestionenzymes) without causing anoikis, cell surface adhesion molecule damage,or other non-anoikis means of cell death. Accordingly, in certainembodiments, after obtaining the sample from the subject, the sample ofviable diseased cells is contacted with a digestion media comprising oneor more enzymatic agents for a period of time, wherein the digestionmedia digests tissue without causing anoikis, cell surface adhesionmolecule damage or non-anoikis means of cell death. The enzymatic agentscan be selected from the following non-limiting examples: collagenasesof any of the known types, hyalronidases, papain, dispase, elastase,trypsin and any combinations thereof. In one embodiment, the digestionmedia comprises a mixture of collagenases and hyaluronidase. When usedin combination, the enzymatic agents may be used in different ratios tooptimize yield and reduce damage to the cells. The tissue specimen maybe placed in a digestion media comprised of any of the agents above fordifferent periods of time, including minutes up to several days. In mostpreferred embodiments, the enzymatic agents are selected to limit theexposure time of the cells to the enzymes and optimize the yield of aheterogenous population of viable cells.

In other embodiments, the digestion media is supplemented with at leastone anoikis inhibitor. A preferred embodiment is to supplement thedigestion media with at least one anoikis inhibitor directed atintrinsic anoikis pathway induction and at least one inhibitor directedat extrinsic pathway induction. The digestion media also can besupplemented with at least one anoikis inhibitor. A further embodimentis to use any anoikis inhibitor or combination of anoikis inhibitors ona continuous or intermittent basis during the extraction process. In oneembodiment, after obtaining the tissue sample from the subject, thecells are cultured in a media containing at least one digestion enzyme(e.g., collagenases and hyaluronidase) and containing at least oneanoikis inhibitor, preferably under conditions of greater than 2% andless than 20% oxygen (more preferably at 6-17% oxygen, more preferablyat 10% oxygen). The cells are cultured in this media for a period oftime (e.g., at least one hour, at least two hours, at least three hoursor for one to five hours or for three hours), and then the cells arecultured in the same media (i.e., comprising anoikis inhibitor(s)) butwithout the digestion enzymes.

Determination that the diseased cells of interest are present whenpracticing the composition or methods of the present invention may bemade by examining expression of markers that identify particular celltypes. Non-limiting examples of markers for cell type identificationinclude those listed in Table 2 below. Use of one or more markersspecific for the general cell type or for the disease of interest,keeping in mind that there is heterogeneity of the sample in many cases,is most desirable. Marker expression characterization, quantification,or presence, can be made by examining the protein, mRNA level, othersmaller RNA levels (e.g. siRNA, lncRNA, microRNA, regulatory RNA) by forexamples FACS, quantitative or other PCR methods, mass spectroscopy, orpolyacrylamide gels.

TABLE 2 Exemplary Markers for Cell Identification in Primary CellSamples Marker Name Cell Type Marker Name Cell Type ESR1 diseaseCD49F/ITGA6 Stem, epithelial PGR disease Claudin4 Stem, HER2 diseasemesenchymal Keratins 8, 18, 19 Luminal epithelial epithelialbeta-catenin MME/CD10 Luminal epithelial Keratins 5, 14 Basal epithelialCYP2b7P1, TFF1, AGR3, ER+ breast disease E-cadherin, epithelial FOXA1epithelial, disease EpCam Smooth muscle actin, CALLA Myo-epithelial ErbBfamily disease A2ML, FABP7, ACE2, Triple negative IGFr, c-myc, diseaseHORMAD1 cancer or disease VEGFr TFF1 disease K19, CD44 Stem, disease

Transformation of cells may be desirable for working with certainprimary cell materials to meet other research or commercial goalsunrelated to the present invention. Since transformation of primarycells potentially creates a cell state that is not representative of thein vivo tumor functional activity, the present invention excludes celltransformation that modifies the genetic, epi-genetic, or diseasefunction of the cells such that the in vivo state is not present.

Additionally, tumorigenicity in mouse models has become a standard fordetermining the presence of cancer cells in an isolated cell sample.Notwithstanding the considerable effort and knowledge that has beengained through such experimentation, the present invention recognizesthat many intrinsic tumor types have not reached acceptable levels ofreliable recapitulation of the in vivo disease and consistency of growthin the mouse to be useful in clinical practice with patients. Many tumortypes will not grow reliably in mice; the mouse is different from humansin fundamental ways at the cellular level (e.g. critical proteins mayhave the same name in both mammals but have different sequences,structures, functions, and location that change and affect function ofthe cellular pathways); and the amount of time to get sufficient mousexenograft material does not meet the time requirement for usefulclinical test results. As such, xenograft models as means to testing orproviding useful cells for clinical testing lie outside the presentinvention.

In an embodiment, the preparation of the cell sample takes place at thelocation where the clinical testing will be performed (e.g., laboratory,hospital). As such, the cells can be preserved in a well-known transfermedium to bridge the time from removal from the subject to initiation ofthe cell sample preparation methods described herein. In anotherembodiment, the preparation of the cell sample takes place at adifferent location than where the specimen is removed from the patientrequiring shipment of the specimen in a specimen collection kit. Thetime required to ship the specimen to the location where the cell samplewill be prepared can range from 1-36 hours, and more than 36 hours.

Preparation of Culture Media

Culture media for use in the methods of the invention can be prepared bystandard methods in which components are added to a base media at thedesired concentration. The preparation of culture media for use in thepresent invention can be made by the addition of individual componentsin liquid or solid form, in aqueous or organic solutions (preferably atnot more than 0.3% final organic solvent composition). Components may becombined before final preparation of the working medium to make bulletconcentrated stock solutions for storage and for ease and uniformity ofpreparation. In the case of the use of bullet concentrated stocksolutions for the practice of the present composition or methods,reagents are preferably combined by aqueous solubility level, i.e. saltsand more water-soluble components are stored as an aqueous bullet typeand less water-soluble, hydrophobic, components are stored as anethanol, dimethylformamide, and or dimethysulfoxide stock. Thecomposition comprising the stock solutions of non-proteinaceouscomponents may be stored ideally −30° C. to −80° C. in a sealed,light-proof container to prolong their useful life time.

Culture media comprising one or more anoikis inhibitors (optionallyincluding other components as described above) can be furtherconditioned in advance of application onto the sample of viable diseasecells by placement in environmental conditions of 10% O₂, 5% CO₂, 37°C., so as to further reduce the potential anoikis effect on cells.

In all embodiments, the medium is used in a sterile or essentiallyaseptic form. This form may be created by various methods known to thoseskilled in the art but preferably is performed in a manner that isnon-destructive to the composition and or individual components. Forexample, a preferable method is filter sterilization using a lowprotein-binding membrane filter 0.1-1.0 micrometer pore size.

In embodiments, apparent to one of ordinary skill in the art, theconcentration of a given component can be increased or decreased beyondthe range listed in the included tables and the effect of the increasedor decreased concentration can be determined using routine empiricalexperimentation. The optimization of the present media formulations forany specific diseased cell or tissue type can be carried out usingempirical approaches for examples such as titration, addition, deletion,or Design of Experiment (DOE), or combinations of these.

Optimization of Culture Conditions

To optimize the cell preparation conditions, which includes compositionof the media, the concentration of the media components, the length oftime the culture media or atmospheric conditions are used, thestages/steps of processing the conditions are applied, the differenttypes of culture media used, or the type of surface the cells areattached to, a series of experiments can be conducted to evaluate thecell expansion rate, the type of viable cells in the cell sample, theproportion of cell types in the cell sample that result, whether or notthe cells attach to a surface, the viability of the cells, themitochondrial activity of the cells, or any other measurement of cellpreparation results under any combinations of these conditions. Theresults from these experiments can be used to select the cellpreparation conditions that are optimal for the requirements of aparticular clinical test, or disease, or cell type, or tissue type.

Other suitable optimization approaches and analyses are readily apparentto the ordinarily skilled artisan given the guidance herein.

Compositions and Kits

Compositions for use in the methods of the invention, as well as kitsfor practice of the methods of the invention are also encompassed.

In one embodiment, the invention provides a kit suitable for theisolation of viable diseased cells and practice of the methods ofpreparing primary cells. This kit can be provided with instruction forthe environmental conditions described herein and for the instructionfor making any form of the present compositions, including culture mediaand attachment surfaces comprising ECM components. For example, the kitcan provide separated stock components that can be combined and dilutedin suitable aqueous solutions. In certain embodiments, at least some ofthe components of the kit are stored at different temperatures or asliquids or as powders. In certain embodiments, the kit includes coatedsurfaces for culturing the diseased cells on an anoikis-reducingsurface, such as the surfaces described herein comprising ECMcomponents.

In another aspect, the invention provides a cell culture compositioncomprising primary human cancer cells cultured in a media comprising atleast one anoikis inhibitor under conditions comprising greater than 2%and less than 20% oxygen. In one embodiment of the cell culturecomposition, the media comprises at least one inhibitor of an intrinsicanoikis pathway and at least one inhibitor of an extrinsic anoikispathway and wherein the cells are cultured under conditions comprising6-17% oxygen (e.g., 10% oxygen). In another embodiment of the cellculture composition, the media comprises at least one inhibitor of anintrinsic anoikis pathway, at least one inhibitor of an extrinsicanoikis pathway and wherein the cells are cultured under conditionscomprising 6-17% oxygen (e.g., 10% oxygen). In yet other embodiments,the media comprises one or more additional components as described inthe section above on use of multiple culture media in combination.

In another aspect, the invention provides a biosensor surface comprisingprimary human cancer cells attached to the biosensor surface via ahydrated extracellular matrix (ECM), wherein the ECM consists of: (i)fibronectin and collagen; (ii) collagen and laminin 332 (laminin V) or(iii) laminin 332 (laminin V). In one embodiment, the ECM consists offibronectin and collagen, preferably comprising a fibrillic andhydrophilic surface. In another embodiment, the ECM consists of collagenand laminin 332 (laminin V). In yet another embodiment, the ECM consistsof laminin 332 (laminin V). In one embodiment, the hydrated ECM isfolded.

In another aspect, the invention provides a cell culture vessel surfacecomprising primary human cancer cells attached to the cell culturevessel surface via a hydrated extracellular matrix (ECM), wherein theECM consists of: (i) fibronectin and collagen; (ii) collagen and laminin332 (laminin V) or (iii) laminin 332 (laminin V). In one embodiment, theECM consists of fibronectin and collagen, preferably comprising afibrillic and hydrophilic surface. In another embodiment, the ECMconsists of collagen and laminin 332 (laminin V). In yet anotherembodiment, the ECM consists of laminin 332 (laminin V). In oneembodiment, the hydrated ECM is folded.

Clinical Testing

The method of the invention for preparing primary cells for clinicaltesting can optionally include, after the culture step(s), a step ofconducting a clinical test of the sample of cells. Thus, at the propertime before testing, the viable diseased patient cell sample can beremoved from the above-described anti-anoikis preparation conditions inorder to conduct one or more clinical tests on the sample. The clinicaltest(s) can be any clinical test that uses primary cells designed toprovide clinically relevant and/or useful results, including but notlimited to diagnostic tests, genetic tests, and tests to determine theefficacy of particular treatment regimens.

In one embodiment, the clinical test is conducted using a biosensor.Accordingly, the methods of the invention, the sample of viable diseasecells can be attached to a biosensor surface prior to clinical testing(e.g., using the anti-anoikis attachment conditions described herein).Non-limiting examples of biosensor-based assays that can be carriedusing the cultured primary cells provided by the invention includeassays for determining whether an agent(s) causes a change in the celladhesion and signaling pathway activity of the viable disease cells,such as the assays described in U.S. Patent Publications 20130330761 and20150125894 by Laing et al., the entire contents of both of which arespecifically incorporated herein by reference in their entirety.

In one embodiment, a sample of viable diseased cells prepared accordingto any of the culturing methods described herein is evaluated todetermine whether the addition of agents causes a change in the celladhesion and signaling pathway activity of the viable disease cells. Incertain embodiments, these cell preparation methods are combined with amethod of evaluating whether a first agent that is a targetedtherapeutic has an effect on a signaling pathway it is intended toaddress in order to determine whether the targeted therapeutic isfunctional in the subject's cancer cells. Such a method can comprise:

culturing the sample of viable cancer cells obtained from the subject ina media free of serum, wherein the media can further contain any of theanti-anoikis components or conditions described herein;

contacting the sample with a first agent and with a second agent that isknown to selectively affect the same signaling pathway the first agentis intended to address, so as to upregulate or down-regulate thesignaling pathway as measured by an effect on cell adhesion orattachment, to produce a sample contacted with both the first agent andthe second agent;

continuously measuring cell adhesion or attachment of viable cells inthe sample contacted with both the first agent and the second agent,relative to a sample of viable cancer cells obtained from the subjectwhich sample is contacted with the first agent or the second agentalone;

determining by mathematical analysis of the continuous measurementswhether a change in cell adhesion or attachment has occurred in thesample contacted with both the first agent and the second agent, ascompared to the sample contacted with the first agent or the secondagent alone; and

selecting the first agent for therapeutic use in the subject wherein thefirst agent, in combination with the second agent, causes a change incell adhesion or attachment, as compared to the first or second agentalone, indicating the first agent is functional in the cell signalingpathway of the subject's cancer cells.

In other embodiments, these cell preparation methods are combined with amethod of evaluating whether a therapeutic that targets a HER familyreceptor is functional in a sample of viable cancer cells obtained froma subject. Such an evaluation method can comprise:

culturing the sample of viable cancer cells obtained from the subject ina media free of serum and growth factors;

contacting (1) a first portion of the sample with a therapeutictargeting a HER family receptor and with neuregulin, and/or (2)contacting a second portion of the sample with a therapeutic targeting aHER family receptor and with an epidermal growth factor;

continuously measuring cell adhesion or attachment of viable cells (1)in the first portion of the sample contacted with both the therapeuticand neuregulin, relative to a sample of viable cancer cells obtainedfrom the subject which sample is contacted with the therapeutic orneuregulin alone, and/or (2) in the second portion of the samplecontacted with both the therapeutic and an epidermal growth factor,relative to a sample of viable cancer cells obtained from the subjectwhich sample is contacted with the therapeutic or an epidermal growthfactor alone;

determining by mathematical analysis of the continuous measurementswhether a change in cell adhesion or attachment has occurred (1) in thefirst portion contacted with both the therapeutic and neuregulin, ascompared to the sample contacted with the therapeutic or neuregulinalone, and/or (2) in the second portion contacted with both thetherapeutic and an epidermal growth factor, as compared to the samplecontacted with the therapeutic or an epidermal growth factor alone; and

selecting the therapeutic to treat the subject wherein a change in celladhesion or attachment occurs (1) in the first portion, as compared tothe therapeutic or neuregulin alone, and/or (2) in the second portion,as compared to the therapeutic or an epidermal growth factor alone,indicating the therapeutic is functional in the subject's cancer cells.

In embodiments, a sample of viable diseased cells prepared according toany of the culturing methods described herein are evaluated to determinewhether the addition of an agent(s) causes a change in the cell adhesionand signaling pathway activity of the viable disease cell. In certainembodiments, these cell preparation methods are combined with a methodof evaluating whether a first agent that is a targeted therapeutic hasan effect on a signaling pathway it is intended to address in order todetermine whether the targeted therapeutic is functional in thesubject's cancer cells. Such a method can comprise:

culturing the sample of viable cancer cells obtained from the subject ina media free of serum and containing selected natural agents or factorsknown to affect cell signaling function that may lead to sensitivity orresistance to the targeted therapeutic or the second agent;

contacting the sample with a first agent and with a second agent that isknown to selectively affect the same signaling pathway the first agentis intended to address, so as to upregulate or down-regulate thesignaling pathway as measured by an effect on cell adhesion orattachment, to produce a sample contacted with both the first agent andthe second agent;

continuously measuring cell adhesion or attachment of viable cells inthe sample contacted with both the first agent and the second agent,relative to a sample of viable cancer cells obtained from the subjectwhich sample is contacted with the first agent or the second agentalone;

determining by mathematical analysis of the continuous measurementswhether a change in cell adhesion or attachment has occurred in thesample contacted with both the first agent and the second agent, ascompared to the sample contacted with the first agent or the secondagent alone; and

selecting the first agent for therapeutic use in the subject wherein thefirst agent, in combination with the second agent, causes a change incell adhesion or attachment, as compared to the first or second agentalone, indicating the first agent is functional in the cell signalingpathway of the subject's cancer cells in the presence of the resistanceor sensitivity agent or factor.

Clinical testing carried out using primary cells prepared according tothe methods described herein, and/or using the compositions describedherein (e.g., primary cell cultures, cell culture vessel surfaces,biosensor surfaces) can be used for diagnostic purposes, e.g., todiagnose whether a subject from whom the primary cell sample is obtainedhas a particular disease or disorder (e.g., cancer, an autoimmunedisease etc.), wherein the diagnostic criteria based on the outcome ofthe clinical testing has been determined.

Furthermore, the subject from whom the primary cell sample is obtainedcan be treated based on the outcome of the clinical testing (e.g., atreatment regimen can be selected based on the outcome of the clinicaltesting). For example, in one embodiment, the cell preparation methodsand compositions described herein are combined with a method of treatinga human subject diagnosed with cancer, wherein the selected treatmentregimen is based on the outcome of clinical testing using the cellpreparation methods and compositions described herein. In oneembodiment, this method of treating a human subject diagnosed withcancer comprises:

-   -   administering to the subject a first agent that is a targeted        therapeutic that has been determined to be therapeutically        active in the signaling pathway it is intended to address in the        subject's cancer cells, by a method comprising:    -   culturing a sample comprising of viable cancer cells (e.g.,        primary and/or metastatic cancer cells) obtained from the        subject (e.g., according to the culturing methods described        herein);    -   contacting the sample with the first agent and with a second        agent that is known to selectively affect the same signaling        pathway the first agent is intended to address, so as to        upregulate or downregulate the signaling pathway as measured by        an effect on cell adhesion or attachment, to produce a sample        contacted with both the first agent and the second agent;

continuously measuring cell adhesion or attachment of viable primary ormetastatic cancer cells in the sample contacted with both the firstagent and the second agent, relative to a sample of viable cancer cellsobtained from the subject which sample is contacted with the first agentor the second agent alone (e.g., using a biosensor, wherein the viablecells are attached to the biosensor surface according to the methodsdescribed herein);

determining by mathematical analysis of the continuous measurements anoutput value, expressed as a percentage, that characterizes whether achange in cell adhesion or attachment has occurred in the samplecontacted with both the first agent and the second agent, as compared tothe sample contacted with the first agent or the second agent alone; and

administering the first agent to the subject wherein the output valuethat characterizes the change in cell adhesion or attachment is equal toor greater than a threshold value (e.g., 50%), indicating the firstagent is therapeutically active in the cell signaling pathway of thesubject's cancer cells.

Definitions

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. If a definition set forth inthis section is contrary to or otherwise inconsistent with a definitionset forth in the patents, applications, published applications and otherpublications that are herein incorporated by reference, the definitionset forth in this section prevails over the definition that isincorporated herein by reference. The following terms, as used herein,are intended to have the following definitions.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10%. In one aspect, the term “about” meansplus or minus 20% of the numerical value of the number with which it isbeing used. Therefore, about 50% means in the range of 45%-55%.Numerical ranges recited herein by endpoints include all numbers andfractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.90, 4, and 5).

The term “activator,” “activate,” or “perturbant,” “perturb,”“perturbation” in conjunction with respect to cells refer to thespecific subject or activity of physiologic manipulation of a cell usingreagents, organic molecules, signaling factors, biochemicals, nucleicacids, or proteins that have an effect on cells well known to thosepracticed in the art. The effect refers to any modulation of cellularphysiologic activity and may include but not be limited to up ordown-regulation.

The term “anoikis” refers to an activity leading to quiescence,senescence, and eventually can lead to cell death that is induced bysignaling of adhesion molecules of anchorage-dependent cells as they aredetaching from the surrounding extracellular matrix (ECM) andneighboring cells. Usually cells stay close to the tissue to which theybelong since the communication between proximal cells as well as betweencells and ECM provide essential signals for regulating growth andsurvival. When cells are detached from their native ECM or theirneighbors, there is a loss of normal cell-matrix and cell-cellinteractions, and the cells will initiate anoikis signaling. The presentinvention is aimed at preventing anoikis signaling of primary cells andanoikis-related activities of primary patient diseased cells such thatsaid cells may be used in a clinical test of the patient's functionalcells that may rely upon measuring response to perturbants viaadhesion-related mediated signaling.

The term “antibiotic” refers to chemical substances natural or syntheticof relatively low molecular weight that inhibit growth of non-mammaliancells. First discovered naturally where they were produced by variousspecies of microorganisms, such as bacteria, actinomycetes, and fungi,they may be synthesized chemically, or natural compounds may bechemically modified to produce semi synthetic antibiotics. The majorclasses of antibiotics are: (1) beta-lactams, including the penicillins,cephalosporins and monobactams; (2) aminoglycosides, e.g., gentamicin,tobramycin, netilmycin, and amikacin; (3) tetracyclines; (4)sulfonamides and trimethoprim; (5) fluoroquinolones, e.g.,ciprofloxacin, norfloxacin, and ofloxacin; (6) vancomycin; (7)macrolides, which include for example, erythromycin, azithromycin, andclarithromycin; and (8) other antibiotics, e.g., the polymyxins,chloramphenicol and the lincosamides. Commercially available mixturesare included (e.g. P/S/A—penicillin-streptomycin-amphotericin B, alsoknown as antibiotic-antimycotic solution).

The term “apoptosis” refers to programmed cell death and is a keyregulatory system of physiological growth control and regulation oftissue homeostasis. Apoptosis plays a critical role in the regulation oftumor formation. Many anticancer therapies are designed to activateapoptosis signal transduction pathways in cancer cells Apoptosis can bea stage after anoikis in some cells.

The term “anoikis inhibitor” refers to molecules or conditions thateither inhibit pro-anoikis function or allow anti-anoikis activation ofintrinsic or extrinsic signal transduction pathways and thereby suppressanoikis. Preferred inhibitors in the present invention are reversibleafter a period of time. A reversible inhibitor in this case involvesinhibition caused by application of a reagent whose effect on anoikis isnot sustained upon removal of the reagent. Reversible inhibitors aredesirable so that patient samples are returned to their in-vivo-likestate before testing.

The term “assay” or “assaying” refers to an analysis to determine, forexample, the presence, absence, quantity, extent, kinetics, dynamics, ortype of a target, such as a cell's optical or bioimpedance response uponstimulation with exogenous stimuli (e.g., therapeutic agent).

The terms “attach,” or “attachment,” refer to, for example, a surfacemodifier substance, a cell, a ligand candidate compound, and likeentities of the disclosure, connected to a surface, such as by physicalabsorption, chemical bonding, chemical attraction, and like processes,or combinations thereof. Particularly, “cell attachment,” “celladhesion,” or “cell sample attachment” refer to the binding of cellstogether or interacting to a surface, such as by culturing, orinteracting with a cell anchoring material, or the like.

The term “attachment pattern” refers to observable traits orcharacteristics of a cell or cell sample's connection to a surface. Anattachment pattern can be quantitative, e.g., number of attachmentsites. An attachment pattern can also be qualitative, e.g., preferredmolecular site of attachment to a hydrated extracellular matrix.

The term “basal morphology” refers to the form and structure of a cellor cell sample prior to the introduction of an agent or stimulus.

The term “baseline measurement” refers to a physiologic beginning pointfor a set of cells to be tested and is based on an evaluation ofmeasurements over a period of time before drug is added.

The term “biosensor” refers to a device that measures an analyte or achange in an analyte or physiologic condition of a cell. In embodiments,the biosensor typically contains three parts: a biological component orelement that binds or recognizes the analyte (including non-limitingexamples such as extracellular matrix, cell signaling molecule, or cellproliferation, tissue, cells, metabolites, catabolites, biomolecules,ions, oxygen, carbon dioxide, carbohydrates, proteins etc.), a detectorelement (operating in a physicochemical manner such as optical,piezoelectric, electrochemical, thermometric, or magnetic), and atransducer associated with both components. The term “biosensor”encompasses optical biosensors and impedance biosensors. The term“optical biosensor” refers to a device that measures fluorescence,absorption, transmittance, density, refractive index, and reflection oflight. In embodiments, an optical biosensor can comprise an opticaltransducer for converting a molecular recognition or molecularstimulation event in a living cell, a pathogen, or combinations thereofinto a quantifiable signal. Additionally, embodiments could include aphotonic crystal device, an optical waveguide device, and a surfaceplasmon resonance device. The term “impedance biosensor” refers to adevice that measures complex impedance changes (delta Z, or dZ) of livepatient cells where impedance (Z) is related to the ratio of voltage tocurrent as described by Ohm's law (Z=V/I). It is sensitive to the localionic environment at the electrode interface with the cells and detectsthese changes as a function of voltage and current fluctuations.Physiologic changes of the cells as a result of normal function orperturbation thereof result in quantifiable changes to the flow ofcurrent around the electrodes and influence the magnitude andcharacteristics of the signal measured. In embodiments, an impedancebiosensor can comprise electrodes or an electrical circuit forconverting a molecular recognition or molecular stimulation event in aliving cell, a pathogen, or combinations thereof into a quantifiablesignal. In embodiments, an ISFET biosensor can comprise an ion selectivefield effect electrical transducer for converting an analyte recognitionor cellular stimulation event in a living cell, a pathogen, orcombinations thereof into a quantifiable signal. When an analyteconcentration in an ISFET biosensor changes, the current in thetransistor changes, which creates a quantification signal.

The term “cell adhesion” refers to the binding of a cell to anothercell, to an extracellular matrix component, or to a surface (e.g.,microtiter plate).

The term “cell proliferation” refers to an increase in the number ofcells as a result of cell growth and cell division.

The term “cell sample” refers to cells isolated from a particularsubject, where the cells are isolated from a subject's biologicalfluids, excretions, or tissues. Cells isolated from tissue can includetumor cells. Cells isolated from tissue include homogenized tissue, andcellular extracts, and combinations thereof. Cell samples includeisolation from, but are not limited to, breast tissue, stomach tissue,colon/intestinal tissue, lung tissue, head tissue, neck tissue, parotidtissue, ovarian tissue, uterine tissue, cervical tissue, prostatetissue, pancreatic tissue, kidney/renal tissue, or bone and/or bonemarrow, blood, blood serum, blood plasma, urine, semen, seminal fluid,seminal plasma, prostatic fluid, pre-ejaculatory fluid (Cowper's fluid),excreta, tears, saliva, sweat, biopsy of any practical form, ascites,surgical resection, cerebrospinal fluid, lymph, marrow, or hair.

The term “cell signaling” refers to the intracellular or intercellulartransfer of information. Cells signaling can be achieved by directcontact between cells, between cells and ECM, or by the release of asubstance from one cell that is taken up by another cell. Intercellularsignaling can occur via an interaction between two molecules (e.g., aligand and a receptor). Receptor binding can trigger a cascade ofintracellular signaling (e.g., initiation of biochemical changes withinthe cell or modification of the membrane potential).

The term “cell survival” refers to the viability of a cell characterizedby the capacity to perform certain functions such as metabolism, growth,movement, reproduction, some form of responsiveness, and adaptability.

The term “chemically defined” means the structures, chemical formula,and the composition and percentage of the various individual componentswithin a chemical composition are known or can be defined. To this end,in the present invention the medium does not contain any animal serumtypically prepared for tissue culture purposes. Nor does it contain anytissue extracts with unknown/undefined chemical components. Allessential components necessary to support the desiredgrowth/proliferation of a heterogenous population of diseased cell typesfrom individual patients are chemically defined.

The term “cytoskeletal organization” refers to the arrangement of theinternal scaffold of a cell. A cell's cytoskeleton comprises filamentsthat serve to support cytoplasmic or membrane elements and/orintracellular organelles. The cytoskeleton also helps to maintain theshape of a cell.

The terms “diseased cell sample” or “diseased cell population” or“diseased cell” refer to a mixture of diseased cells, especiallyepithelial cells that are extracted from an individual patient's tumoror tumors or diseased tissue or fluid. The disease cell sample may befrom tissue or fluid that is characterized by abnormal growth. Thesample of cells is heterogeneous in the composition of particularsub-types and can be comprised of, for example, luminal, myo-, basal,stem, cells on the epithelial mesenchymal continuum, differentiated orundifferentiated, or especially mixtures of all the above where theyrepresent the in vivo condition of a patient's tumor. It is recognizedthat particular cancer intrinsic subtypes may have differentialpercentages of certain types of diseased cells (e.g. ER+ breast canceris known as a luminal epithelial cancer and thus may have a highproportion of extracted cells of luminal lineage and a lesser percentageof other epithelial types. Another example—triple negative breast canceris known as a basal epithelial cancer and thus may have a highproportion of extracted diseased cells of basal lineage and a lesserpercentage of other epithelial types). The sample may be comprisedespecially of viable, or replication competent, or dividing, ornon-dividing, or at any point or combinations of points in the cellcycle. Cells may be entering or not entering anoikis, or fully engagedat any level in anoikis. Samples of cells may be selected on the basisof their disease pathway activity.

The terms “extracellular matrix component” or “ECM component” refer to amolecule that occurs in the extracellular matrix of an animal. Themolecule can be a component of an extracellular matrix from any speciesand from any tissue type. Non-limiting examples of extracellular matrixcomponents include laminins, collagens, fibronectins, otherglycoproteins, peptides, glycosaminoglycans, proteoglycans, etc.Extracellular matrix components can also include growth factors.

The term “extracellular matrix coating” refers to a coating on a cellculture surface that comprises one or more molecules that are naturallyoccurring biomolecules or biochemicals, or biochemicals derived from orbased on one or more naturally occurring biomolecules or biochemicals,that may be found in an extracellular matrix. For example, anextracellular matrix coating can comprise e.g., fibronectin, collagens,laminins, other glycoproteins, peptides, glycosaminoglycans,proteoglycans, vitronectin, IntercellularCAMs, VascularCAMs, MAdCAMs),or a derivative thereof, or can comprise a biochemical such aspolylysine or polyornithine, which are polymeric molecules based on thenaturally occurring biochemicals lysine and ornithine. Polymericmolecules based on naturally occurring biochemicals such as amino acidscan use isomers or enantiomers of the naturally-occurring biochemicals.Coatings can also include cell surface receptor or cell surface cognatebinding proteins or proteins with affinity for said cell surfaceproteins. Ideally an extracellular matrix used for coating is selectedfrom the available genus and species by matching the ECM molecule to aspecific cell type and signaling pathway.

The term “extrinsic anoikis” refers to activity associated with theextrinsic signal transduction pathways of a viable cell. Extrinsicpathways can be activated by death receptors on the plasma membrane suchas for example tumor necrosis factor receptor 1 (TNFR1) and Fas/CD95. Asligands bind to these receptors, the death inducing signaling complex(DISC) is formed leading to initiation of enzyme cascades of which thecaspase cascade is a member and further downstream anoikis events.

The term “extrinsic anoikis inhibitor” refers to an agent that inhibitsanoikis by affecting (e.g., interfering with) the extrinsic anoikispathway, for example an agent that inhibits the activity of a deathreceptor (e.g., TNFR1, ALK5/TGFBR1, Fas/CD95) on the plasma membrane.Non-limiting examples of extrinsic anoikis inhibitors include agentsthat bind to or otherwise inhibit the activity of the death receptor,such as antibodies and Ig fusion proteins (e.g., directed against TNFR1,ALK5/TGFBR1 or Fas/CD95), as well as agents that bind to or otherwiseinhibit the activity of a ligand for the death receptor, such asantibodies and Ig fusion proteins (e.g., directed against TFNα, TGFβ orFasL). Other non-limiting examples of extrinsic anoikis inhibitorsinclude small molecule agents that inhibit signaling through a deathreceptor (e.g., inhibit signaling through TNFR1, ALK5/TGFBR1 orFas/CD95), WNT signaling agonists. Other non-limiting examples ofextrinsic anoikis inhibitors include integrin stabilizers and integrinligands that inhibit anoikis.

The term “folded extracellular matrix” refers to an ECM that is areproducible, ordered, and structured arrangement of proteincomponent(s) in their natural or native states comprising the ECM.

The term “healthy cell sample” refers to a cell sample wherein the cellsdo not have or are extracted from a tissue that does not have thedisease that is being tested. For example, when a particular subject isbeing tested for the effects of a therapeutic agent against thesubject's breast cancer, non-cancerous cells or cells from non-breasttissue are considered “healthy”. The term “healthy cell sample” is not adetermination or reflection upon the whole health status of the subject.

The term “hydrated extracellular matrix” refers to extracellular matrixprepared from extracellular matrix components that has been applied to asurface intended for human cells to attach and which, after applicationto a surface, retains sufficient water such that the ECM is fully wettedand is never allowed to dehydrate prior to use.

The term “hypoxia” refers to cell sample conditions that are less than20.094% partial pressure of oxygen (so called normoxic). The partialpressure of oxygen in the atmosphere is 20.094% at sea level and mostcancer cells are reported to experience less than 2% partial pressure ofoxygen in vivo. Very low oxygen levels and high oxygen levels such asnormoxia, 20% oxygen, are reportedly cause for most cells and especiallyprimary cells to enter anoikis.

The term “intrinsic anoikis” refers to activity associated with theintrinsic signal transduction pathways of a viable cell. Intrinsicanoikis pathways are characterized by permeabilization of themitochondrial membrane and release of cytochrome c into the cytoplasm.Cytochrome c can then form a multi-protein complex and initiatesactivation of the caspase cascade and further downstream anoikis events.

The term “intrinsic anoikis inhibitor” refers to an agent that inhibitsanoikis by affecting (e.g., interfering with) the intrinsic pathway, forexample an agent that inhibits the permeabilization of the mitochondrialmembrane and/or release of cytochrome C into the cytoplasm. Non-limitingexamples of intrinsic anoikis inhibitors include stress inhibitors, suchas redox buffering agents and reactive oxygen species inhibitors.

The term “sample” refers to anything which may contain a moiety to beisolated, manipulated, measured, quantified, detected or analyzed usingapparatuses, microplates or methods in the present disclosure. Thesample may be a biological sample, such as a biological fluid or abiological tissue. Examples of biological fluids include suspension ofcells in a medium such as cell culture medium, urine, blood, plasma,serum, saliva, semen, stool, sputum, cerebral spinal fluid, tears,mucus, amniotic fluid or the like. Biological tissues are aggregates ofcells, usually of a particular kind together with their intercellularsubstance that form one of the structural materials of a human, animal,plant, bacterial, fungal or viral structure, including connective,epithelium, muscle and nerve tissues. Examples of biological tissuesalso include organs, tumors, lymph nodes, arteries and individualcell(s). The biological samples may further include cell suspensions,solutions containing biological molecules (e.g. proteins, enzymes,nucleic acids, carbohydrates, chemical molecules binding to biologicalmolecules).

The terms “serum-free” or “media free of serum” refer to cell culturemedium that contains essentially no serum or tissue extracts. In certainembodiments, there is 0% (completely free), or less than about 0.001%,0.005%, 0.01%, 0.10%, or 1.0% total serum in the medium. Examples oftypes of serums include: various forms of bovine serum (calf serum,fetal bovine serum, bovine calf serum, donor bovine calf serum, definedfetal bovine serum, newborn bovine calf serum, etc.), horse serum andhuman serum, dextran extracted serums, heat inactivated serums,gamma-irradiated serums, heat-inactivated-dextran-extracted serums.

The term “shared pathway anoikis inhibitor” refers to an agent thatinhibits anoikis by affecting (e.g., interfering with) a cellularsignaling pathway or component that is shared by the intrinsic andextrinsic anoikis pathways such that the shared inhibitor may beeffective in inhibiting both extrinsic anoikis and intrinsic anoikis.Non-limiting examples of shared pathway anoikis inhibitors includeRho-associated kinase inhibitors, caspase inhibitors, matrixmetalloprotease inhibitors, WNT signaling agonists, and cytochrome Cinhibitors.

The term “substantially free” refers to at least about 90%, preferably95%, 99% or greater percentage isolated from other components. Thecomponents for example may be other cells, reagents, proteins, peptides,compounds or compositions described herein.

The term “synergy” or “synergistic effect” refers to an interaction oftwo or more agents wherein the combined effect of the two or more agentsis greater than the sum of the separate (individual) effects of eachagent.

The terms “targeted pathway drug,” “pathway drug,” or “targeted drug,”refer to any molecule or antibody with therapeutic capacity designed tobind to a specific biomolecule (e.g. protein) involved in a diseaseprocess, thereby regulating its activity.

The term “therapeutic agent” refers to any synthetic or naturallyoccurring biologically active compound or composition of matter which,when administered to an organism (human or nonhuman animal), induces adesired pharmacologic, immunogenic, and/or physiologic effect by localand/or systemic action. The term encompasses those compounds orchemicals traditionally regarded as drugs, vaccines, andbiopharmaceuticals including molecules such as proteins, peptides,hormones, nucleic acids, gene constructs and the like. The agent may bea biologically active agent used in medical, including veterinary,applications and in agriculture, such as with plants, as well as otherareas. The term therapeutic agent also includes without limitation,medicaments; vitamins; mineral supplements; substances used for thetreatment, prevention, diagnosis, cure or mitigation of disease orillness; or substances which affect the structure or function of thebody; or pro-drugs, which become biologically active or more activeafter they have been placed in a predetermined physiologicalenvironment. Therapeutic agents include, but are not limited to,anticancer therapeutics, antipsychotics, anti-inflammatory agents, andantibiotics.

Where ranges are given, endpoints are included. Furthermore, it is to beunderstood that unless otherwise indicated or otherwise evident from thecontext and understanding of one of ordinary skill in the art, valuesthat are expressed as ranges can assume any specific value or subrangewithin the stated ranges in different embodiments of the invention, tothe tenth of the unit of the lower limit of the range, unless thecontext clearly dictates otherwise.

All cited sources, for example, patents, patent publications,references, publications, databases, database entries, and art citedherein, are incorporated into this application by reference in theirentirety, even if not expressly stated in the citation. In case ofconflicting statements of a cited source and the instant application,the statement in the instant application shall control.

Examples

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Discussion of Experimental Design

The methods for obtaining and culturing primary diseased cells asdescribed in the present invention are used to prepare diseased cellsfor a clinical test.

It is commonly understood that biochemical principles of protein bindingand anoikis are universal across different cell types that attach toECM, independent of their tissue of origin. The methods described hereinare thus broadly applicable to all cell types that are attachable to anECM regardless of their tissue of origin.

The three examples provided below demonstrate various embodiments of themethods for preparing primary cells for clinical testing describedherein. In particular, the examples demonstrate the advantages thatoccur when: 1) 10% oxygen conditions, or 2) one or more anoikisinhibitors, or 3) hydrated and folded ECM are used when culturing a cellsample. Each of these three variables are evaluated in isolationrelative to conventional conditions using a “one variable at a time”methodology. When these variables are isolated in the three examples,they are labeled for discussion purposes as the “improved” conditionsand the conditions they are compared to are labeled as the“conventional” condition.

-   -   Example 1—Comparison of culturing methods under different oxygen        conditions    -   Example 2—Comparison of culturing methods with and without one        or more anoikis inhibition molecules    -   Example 3—Comparison of culture methods with and without        hydrated folded ECM        Elements Used in Examples

Tissue: Diseased breast tissue obtained from two different patientspecimens was used in these examples (C54, C517A). Hypertrophic andbreast cancer tissue provides exemplary tissue models since theirmechanisms of anoikis and anoikis regulation are representative of otherdiseased tissue types. In addition, breast cancer accounts for over 20%of all cancers diagnosed annually in the United States, and it is themost common form of cancer in women. Tissue from the patients wereobtained using tissue specimen collection techniques as describedherein. Each cell sample tested was extracted from separate aliquots ofdiseased breast tissue. Each specimen aliquot consisted of 30 milligramsof tissue.

Cells: Diseased epithelial cells extracted from patient breast tissuespecimens were selected for testing. Epithelial cells are an exemplarycell model since the mechanisms of anoikis and anoikis regulation foundin them are representative of other tumor cell types.

Anoikis inhibitors. Three anoikis inhibitor molecules were used in theseexperiments—Rho-Kinase inhibitor, Caspase Inhibitor, and MMP3Inhibitor—alone and in combination. Each of these affect differentanoikis regulatory pathways and their associated function, including howthey are extensively interconnected, regulated through binding, involveenzymatic activities such as protein cleavage, phosphorylation andde-phosphorylation, and control critical cellular functions.

Extracellular matrix (ECM) coating. Two types of ECM coating wereevaluated: 1) hydrated and folded; 2) non-hydrated and non-folded. Eachcoating was comprised of human collagen I and human fibronectin.

Oxygen. Atmospheric conditions with three different oxygenconcentrations were evaluated: 1) 2% oxygen; 2) 10% oxygen; 3) 20%oxygen.

Assessment of Culture Conditions

Two key goals when culturing a diseased cell sample are to expand thesize of the cell colonies and the number of cells available for testing.A set of culture conditions that results in both higher cell count andcell colony size compared to the results from a different set ofconditions is superior.

In each of the three following examples, cell samples were culturedunder identical conditions, except for one variable, and then compared.To compare the effect a specific culture condition variable has on acell sample obtained from the same patient, two different metrics wereused—cell count and cell colony size change. Cell count allows for aside by side comparison of the increase in number of cells producedunder different culture conditions. Cell colony size allows for acomparison of the change in the size of cell colonies produced underdifferent culture conditions. Each metric provides a different insightinto the status of a cell sample after being cultured. Cell countprovides a short-term assessment of the culture conditions whereas cellcolony size change gives an indication about the overall longer-termrobustness of the cell sample.

Each of the three different examples are designed to isolate the effectone of three key variables—atmospheric oxygen concentration, presence orabsence of anoikis inhibitors, and the type of ECM coating—has on a cellsample. In each example, an “improved” condition is tested relative to a“conventional” condition. The improved condition is intended to improvethe results of the cell culture by reducing the deleterious effects ofanoikis on the cell sample. In each example, cell count and cell colonysize is measured after the cell sample has been cultured for a number ofdays to measure the effect of ameliorating anoikis within a cell sample.If one set of conditions results in both higher cell count and cellcolony size, then those conditions would be deemed superior relative toits comparator.

Cell Count. An electronic cell counting instrument, Countess (LifeTechnologies), was used to count the number of cells in a cell sample atthe end of each experiment. In each example, two cell samples derivedfrom two separate tissue aliquots from the same patient are testedside-by-side under two different conditions. Prior to counting, the cellsample was treated with trypan blue to enable distinction between liveand dead cells. Only live cells (those not stained by the trypan blue)were counted. All cell samples were counted in duplicate after five daysin culture and the cell counts and are reported. The cell counts fromeach cell sample cultured side-by-side are compared by calculating therelative percentage difference between the “improved” cell culture andthe “conventional” cell culture. A cell culture condition that yielded ahigher cell number count after five days relative to its comparator wasdeemed superior.

Cell colony size. To quantify the change in the size of cell colonies ina cell sample, an assessment of the size of four cell colonies is madeat a first (early) date after initiation of culturing and compared tothe sizes of those four colonies at a second, later date. The firstimage is taken 48 hours after culture initiation and the second image istaken 48-96 hours later. To measure colony size, images of colonies aremade using a 40× inverted microscope and the area of the cell coloniesis then quantified by determining the number of pixels each colonycovers. The absolute change in the total size of the four coloniesbetween the first and second images is calculated for each cell sampleunder the two different conditions and reported. This change in colonysize is then compared by calculating the relative percentage differencebetween the “improved” cell culture and the “conventional” cell culture.A cell culture condition that yielded a larger change in cell colonysize relative to its comparator was deemed superior.

Example 1: Comparison of Culturing Methods Under Three Different OxygenConditions

In this example, primary cell samples from patient C517 were prepared inan extraction culture media and an initial culture media that containedat least one anoikis inhibitor on a cell culture surface comprised of ahydrated and folded ECM coating under three different oxygenconditions: 1) 10% oxygen conditions (the “improved” condition); 2)hypoxic (2%) oxygen conditions; 3) normoxic (20%) conditions. The 2% and20% oxygen conditions represent the “conventional” condition.

The cell sample for this example was prepared as follows:

A human diseased tissue specimen was obtained and initially prepared byremoving obviously fatty portions and mincing the specimen into 1-4 mmsize pieces. The minced specimen was then separated into aliquots, each30 milligrams in size.

Diseased cells were extracted from the minced specimen sample aliquot bytumbling the sample in an extraction media for at least three hours(“extraction period”) that was serum free and included DMEM/F12 media, amixture of collagenases and hyaluronidase (digestion enzymes), and atleast one anoikis inhibitor molecule. In addition, the media comprisedadditional components, including: epidermal growth factors, estradiol,Ca2+, Mg2+, tri-iodothyronine, tetra-iodothyronine, glutathione,adenine.

The cells extracted from the tissue sample were recovered from theextraction media and plated on a surface comprising a hydrated andfolded extracellular matrix consisting of a combination of collagen andfibronectin.

The cell sample was then cultured in the media used for extraction(described above) but with the digestion enzymes (collagenases andhyaluronidase) removed.

All tissue samples were cultured at 37° C., 5% CO₂, 85% relativehumidity and either 2%, 10%, or 20% oxygen (the variable condition beingevaluated).

The cells were then removed from the culture vessel by treatment with agentle enzyme removal process, followed by washing and transfer to atest vessel.

Results for the three samples cultured are shown in Tables 3 and 4. Ineach table, the “improved” condition (10% oxygen) is compared to theconventional condition (2% or 20% oxygen). Cell count and cell colonysize change and the percent difference between the optimized conditionand the conventional condition is reported. Cell colony size change isreported in pixels.

TABLE 3 10% vs. 2% Oxygen Difference 10% O₂ 2% O₂ (Improved vs. Metric(Improved) (Conventional) Conventional) Cell count (live only) 141,2256,818 1971% Cell colony size change 552,025 62,442 784% (pixels)

Both cell count and cell colony size were dramatically higher under the10% oxygen conditions compared to the 2% conditions. These demonstratethe superiority of using 10% oxygen versus 2% oxygen (hypoxia)conditions when culturing live cell samples.

TABLE 4 10% vs. 20% Oxygen Difference 10% O₂ 20% O₂ (Improved vs. Metric(Improved) (Conventional) Conventional) Cell count (live only) 141,22519,988 606% Cell colony size change 552,025 75,087 635% (pixels)

Both cell count and cell colony size were dramatically higher under the10% oxygen conditions compared to the 20% conditions. These results thusdemonstrate the superiority of using 10% oxygen versus 20% oxygen(normoxic) conditions when culturing live cell samples.

In summary, these examples demonstrate the superiority of culturingcells in atmospheric conditions that are below normoxic conditions (20%)and above hypoxic conditions (2%). The results for 2% and 20% conditionswere comparable, which suggests that cell sample results improve as theoxygen concentration converges on 10%. Thus, the ordinarily skilledartisan will readily appreciate the results presented herein that anyoxygen concentration higher than 2% and lower than 20% is superior tothe hypoxic (2%) or normoxic (20%) oxygen concentrations typically usedto culture cells.

Example 2: Comparison of Culturing Methods with and without One or MoreAnoikis Inhibition Molecules

In this example, primary cell samples from two patients, C54 and C517,were tested to isolate the effect anoikis inhibitor molecules have oncell count and cell colony size. Each cell sample was cultured on a cellculture surface comprised of a hydrated and folded ECM coating and under10% oxygen conditions. The samples were cultured in one of fivedifferent culture medias differentiated only by the absence of anyanoikis inhibitor molecules or the presence of one of three differentanoikis inhibitor molecules alone or in combination, as follows: 1) noanoikis inhibitor molecules; 2) a caspase inhibitor; 3) an MMP3inhibitor; 4) a Rho-associated kinase inhibitor; 5) a combination ofcaspase, MMP3 and Rho-associated kinase inhibitors.

The cell sample for this example was prepared as follows:

A human diseased tissue specimen was obtained and initially prepared byremoving obviously fatty portions and mincing the specimen into 1-4 mmsize pieces. The minced specimen was then separated into aliquots, each30 milligrams in size.

Diseased cells were extracted from the minced specimen sample aliquot bytumbling the sample in an extraction media for at least three hours(“extraction period”) that was serum free and included DMEM/F12 media, amixture of collagenases and hyaluronidase (digestion enzymes), andeither: 1) no anoikis inhibitors; 2) a caspase inhibitor; 3) an MMP3inhibitor; 4) a Rho-associated kinase inhibitor; or 5) a combination ofcaspase, MMP3 and Rho-associated kinase inhibitors (the variablecondition being evaluated). In addition, the media in each of the fivedifferent alternative medias evaluated comprised additional components,including: epidermal growth factors, estradiol, Ca2+, Mg2+,tri-iodothyronine, tetra-iodothyronine, glutathione, adenine.

The cells extracted from the tissue sample were recovered from theextraction media and plated on a surface comprising a hydrated andfolded extracellular matrix consisting of a combination of collagen andfibronectin.

The cell sample was then cultured in the media used for extraction(described above) but with the digestion enzymes (collagenases andhyaluronidase) removed.

All tissue samples were cultured at 37° C., 5% CO₂, 85% and 10% oxygen.

The cells were then removed from the culture vessel by treatment with agentle enzyme removal process, followed by washing and transfer to atest vessel.

Results for the three samples cultured are shown in Tables 5-8. In eachtable, the “improved” condition (one or more anoikis inhibitor) iscompared to the conventional condition (no anoikis inhibitor). Cellcount and cell colony size change and the percent difference between theoptimized condition and the conventional condition is reported. Cellcolony size change is reported in pixels.

TABLE 5 Rho-Kinase anoikis inhibitor vs. no anoikis inhibitor molecule(Patient C517) RhoKinase Difference Inhibitor No Inhibitor (Improved vs.Metric (Improved) (Conventional) Conventional) Cell count (live only)78,750 51,450 53% Cell colony size change 502,999 149,309 237% (pixels)

TABLE 6 Caspase anoikis inhibitor vs. no inhibitor (Patient C54) CaspaseDifference Inhibitor No Inhibitor (Improved vs. Metric (Improved)(Conventional) Conventional) Cell count (live only) 69,975 63,750 10%Cell colony size change 194,095 118,754 63% (pixels)

TABLE 7 MMP3 anoikis inhibitor vs. no inhibitor (Patient C54) MMP3Difference Inhibitor No Inhibitor (Improved vs. Metric (Improved)(Conventional) Conventional) Cell count (live only) 137,700 63,750 116%Cell colony size change 336,700 118,754 184% (pixels)

TABLE 8 Three Anoikis inhibitors vs. no inhibitor molecule (PatientC517) 3 Anoikis Difference Inhibitors No Inhibitor (Improved vs. Metric(Improved) (Conventional) Conventional) Cell count (live only) 177,75051,450 245% Cell colony size change 552,025 149,309 270% (pixels)

Both cell count and cell colony size were higher in each cell samplethat was prepared in a culture media containing one or more anoikisinhibitor molecules compared to the cell sample comparator that wascultured with no anoikis inhibitor. These results thus demonstrate thesuperiority of using one or more anoikis inhibitors in a culture mediawhen culturing live cell samples. In addition, since three differentanoikis inhibitor molecules affecting three different anoikis relatedpathways were evaluated, and each found to produce superior results,this example demonstrates that the advantages of using an anoikisinhibitor molecules are general in nature, and not restricted to onespecific molecule or class of anoikis inhibitors.

Example 3: Comparison of Culture Methods with and without HydratedFolded ECM

In this example, primary cell samples from Patient C54 were tested toisolate the effect that coating a cell culture surface with hydrated andfolded ECM has on cell count and cell colony size. Each cell sample wascultured under 10% oxygen conditions in a culture media containing atleast one anoikis inhibitor in a cell culture vessel coated eitherwith: 1) a hydrated and folded ECM; or 2) a non-hydrated and unfoldedECM.

The cell sample for this example was prepared as follows:

A human tumor tissue specimen was obtained and initially prepared byremoving obviously fatty portions and mincing the specimen into 1-4 mmsize pieces. The minced specimen was then separated into aliquots, each30 milligrams in size.

Tumor cells were extracted from the minced specimen sample aliquot bytumbling the sample in an extraction media for at least three hours(“extraction period”) that was serum free and included DMEM/F12 media, amixture of collagenases and hyaluronidase (digestion enzymes), and atleast one anoikis inhibitor molecule. In addition, the media comprisedadditional components, including: epidermal growth factors, estradiol,Ca2+, Mg2+, tri-iodothyronine, tetra-iodothyronine, glutathione,adenine.

The cells extracted from the tissue sample were recovered from theextraction media and plated on a surface comprising either: 1) ahydrated and folded extracellular matrix consisting of a combination ofcollagen and fibronectin; or 2) a non-hydrated and not foldedextracellular matrix (the variable condition being evaluated).

The cell sample was then cultured in the media used for extraction(described above) but with the digestion enzymes (collagenases andhyaluronidase) removed.

All tissue samples were cultured at 37° C., 5% CO₂, 85% relativehumidity and 10% oxygen.

The cells were then removed from the culture vessel by treatment with agentle enzyme removal process, followed by washing and transfer to atest vessel.

Results for the two samples cultured are shown in Table 9 below. In thistable, the “improved” condition (hydrated and folded ECM) is compared tothe conventional condition (non-hydrated and not folded ECM). Cell countand cell colony size change and the percent difference between theoptimized condition and the conventional condition is reported. Cellcolony size change is reported in pixels.

TABLE 9 Hydrated/folded ECM vs non-hydrated/not folder ECM Hydrated &Non-hydrated Difference Folded & Not folded (Improved vs. Metric(Improved) (Conventional) Conventional) Cell count (live only) 69,97539,150 79% Cell colony size change 194,095 50,103 287% (pixels)

Both cell count and cell colony size are higher in each cell sample thatwas prepared using hydrated and folded ECM compared to one prepared withnon-hydrated and not folded ECM. These results thus demonstrate thesuperiority of using a cell culture vessel coated with a hydrated andfolded ECM when culturing live cell samples.

The invention claimed is:
 1. A method of increasing the cell count andcell colony size of viable cancer cells obtained from a human, themethod comprising: culturing the viable cancer cells obtained from thehuman subject in a medium comprising at least one caspase inhibitor, atleast one MMP3 inhibitor and at least one Rho-associated kinaseinhibitor under conditions comprising greater than 2% and less than 20%oxygen for at least 48 hours; and attaching the cultured cancer cells toa surface comprising a hydrated and folded extracellular matrix (ECM).2. The method of claim 1, wherein the viable cancer cells are culturedunder conditions comprising 6-17% oxygen.
 3. The method of claim 1,wherein the cultured cancer cells are transferred to a medium lackingcaspase inhibitors, MMP3 inhibitors and Rho-associated kinase inhibitorsunder conditions comprising 20% oxygen prior to attaching the culturedcancer cells to the surface comprising a hydrated and folded ECM.
 4. Themethod of claim 1, wherein after the cultured cancer cells are attachedto the surface, a clinical test is conducted on the cultured cancercells.
 5. The method of claim 1, wherein the surface is a biosensorsurface or a cell culture vessel surface.
 6. The method of claim 4,wherein a clinical test is conducted on the cells attached to thesurface using a biosensor.
 7. The method of claim 1, which comprises:culturing the viable cancer cells obtained from the human subject in amedium comprising at least one digestion enzyme and at least one caspaseinhibitor, at least one MMP3 inhibitor and at least one Rho-associatedkinase inhibitor under conditions comprising 6-17% oxygen; followed byculturing the viable cancer cells in a medium comprising at least onecaspase inhibitor, at least one MMP3 inhibitor and at least oneRho-associated kinase inhibitor and lacking digestion enzymes underconditions comprising 6-17% oxygen, prior to attaching the culturedcancer cells to the surface comprising a hydrated and folded ECM.
 8. Themethod of claim 1, which comprises: culturing the viable cancer cellsobtained from the human subject in a media comprising at least onecaspase inhibitor, at least one MMP3 inhibitor and at least oneRho-associated kinase inhibitor under conditions comprising 6-17% oxygenon a cell culture vessel surface coated with a hydrated and foldedextracellular matrix (ECM), prior to attaching the cultured cancer cellsto the surface comprising a hydrated and folded ECM.
 9. The method ofclaim 1, wherein: the cultured cancer cells are attached to a biosensorsurface comprising a hydrated and folded extracellular matrix (ECM),wherein the ECM consists of: (i) fibronectin and collagen; (ii) collagenand laminin 332 (laminin V) or (iii) laminin 332 (laminin V).
 10. Amethod of increasing the cell count and cell colony size of viablecancer cells obtained from a human subject for a clinical test using abiosensor, the method comprising: culturing the of viable cancer cellsobtained from the human subject in medium comprising at least onecaspase inhibitor, at least one MMP3 inhibitor and at least oneRho-associated kinase inhibitor under conditions comprising 6-17% oxygenfor at least 48 hours; attaching the cultured cancer cells to abiosensor surface comprising a hydrated and folded extracellular matrix(ECM), wherein the ECM consists of: (i) fibronectin and collagen; (ii)collagen and laminin 332 (laminin V) or (iii) laminin 332 (laminin V);and conducting a clinical test using a biosensor on the cultured cancercells.